Apparatus and method for molecular separation, purification, and sensing

ABSTRACT

Described are devices and methods for forming one or more nanomembranes including electroactive nanomembranes within a nanowell or nanotube, or combinations thereof, in a support material. Nanopores/nanochannels can be formed by the electroactive nanomembrane within corresponding nanowells. The electroactive nanomembrane is capable of controllably altering a dimension, a composition, and/or a variety of properties in response to electrical stimuli. Various embodiments also include devices/systems and methods for using the nanomembrane-containing devices for molecular separation, purification, sensing, etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/642,049, filed Jul. 5, 2017 (now U.S. Pat. No. 10,422,000, issuedSep. 24, 2019), which is a continuation of U.S. patent application Ser.No. 14/714,681, filed May 18, 2015 (now U.S. Pat. No. 9,732,384, issuedAug. 15, 2017), which is a divisional of U.S. application Ser. No.13/437,817, filed Apr. 2, 2012 (now U.S. Pat. No. 9,434,990, issued Sep.6, 2016), the entire disclosures of which are incorporated herein byreference in their entireties.

FIELD OF INVENTION

The embodiments of the invention relate to an apparatus and methods formodulating the dimensionality of nanomembrane(s) and/or nanochannel(s)within nanowell(s) and/or nanotubes in a tunable fashion. The apparatusand methods can be used for a variety molecular analyte isolation anddetection applications, for example to purify and/or quantify analytessuch as proteins, ions, and nucleic acids. The apparatus and methodsalso enable other applications such as nucleic acid sequencing.

BACKGROUND

The technologies for the separation, purification, and sensing ofmolecules has evolved considerably over the past decades. However, thesedevices generally continue to require the use of expensive, fragile,complex to operate, and/or bulky instrumentation such that they cannotreadily be deployed for use in the field, in doctor's offices, or at apatient's bedside. Widespread advances by nanotechnologists and thesilicon manufacturing industries are particularly helping to overcomethese challenges. For example, many chip-based systems are now beingmarketed that enable molecules like proteins, nucleic acids, ions, andsmall molecules to be processed, identified, and quantified.

An ongoing shortcoming, though, is that such chip-based molecularmanipulation systems most often are so specialized that they can only beused for a single purpose: they can quantify DNA but they cannot be usedto purify DNA; they can identify proteins, but their use cannot beextended to identify nucleic acids). In short, a robust, general-purposenanotechnology system that can be used to separate, identify, and/orquantify molecules is currently lacking.

SUMMARY

In accordance with various embodiments, there is provided a method formolecular separation, purification, and sensing. In such a method, oneor more nanowells or nanotubes or combinations thereof may be providedin a support material with each nanotube including one or more sidewallelectrodes and/or each nanowell including one or more sidewallelectrodes and/or one or more bottom electrodes. One or moreelectroactive nanomembranes may also be provided on at least a portionof the one or more sidewall or bottom electrodes or combinationsthereof. A lipid bilayer may be nucleated, stabilized, and/or formed atthe one or more electroactive nanomembranes followed by inserting aprotein nanopore into the lipid bilayer. By applying an ion funnelcurrent or voltage, nucleic acids may be moved towards and through theprotein nanopore. A sequence of the nucleic acids may be derived bycomparing measured signals of a passage of the nucleic acids through theprotein nanopore with predetermined signals.

In embodiments, the one or more nanomembranes may be capable of alteringone or more of a molecular composition, a dimension, or a propertythereof in response to electrical stimuli. For example, the property maybe one or more of hydrophobic, hydrophilic, charged, chemicallyreactive, metal-binding, or metallic property, or combinations thereof.In embodiments, the protein nanopore may include α-hemolysin and/ormycobacterium smegmatis porin A, while the lipid bilayer may besize-restricting to prevent more than one protein nanopore to beinserted in the lipid bilayer. In embodiments, the nucleic acids mayinclude single stranded nucleic acids, double-stranded nucleic acids,partially double-stranded nucleic acids, and/or combinations thereof. Inone embodiment where the nucleic acids are single stranded nucleicacids, one or more complementary nucleic acid oligomers are hybridizedwith the single stranded nucleic acids in a length or under conditionsthat provide for sufficient stability to maintain at least a partiallydouble-stranded form for testing. In one embodiment where the nucleicacids include a strand of RNA for sequencing, by using the disclosedmethod, no reverse transcriptase or optical detection method is used forsequencing the strand of RNA. In one embodiment where the nucleic acidsinclude double stranded DNA, by using the disclosed method, the doublestranded DNA is sequenced without making the double stranded DNAsingle-stranded. In embodiments, the disclosed method may be a signaldetection method using a chloride/silver/silver chloride measuringsystem.

In accordance with various embodiments, there is provided a method formolecular separation, purification, and sensing. In such a method, oneor more nanowells or nanotubes or combinations thereof may be providedin a support material with each nanotube including one or more sidewallelectrodes and/or each nanowell including one or more sidewallelectrodes and/or one or more bottom electrodes. One or moreelectroactive nanomembranes may also be provided on at least a portionof the one or more sidewall or bottom electrodes or combinations thereofto form a nanopore or a nanochannel by the one or more electroactivenanomembranes. Nucleic acids may be placed above the nanopore or thenanochannel, and may be moved towards and through the nanopore or thenanochannel by applying an ion funnel current or voltage to. A sequenceof the nucleic acids may then be derived by comparing measured signalsof a passage of the nucleic acids through the nanopore or nanochannelwith predetermined signals. In embodiments, the one or morenanomembranes may be capable of altering one or more of a molecularcomposition, a dimension, or a property thereof in response toelectrical stimuli. For example, the property may be one or more ofhydrophobic, hydrophilic, charged, chemically reactive, metal-binding,or metallic property, or combinations thereof. In embodiments, thenucleic acids may include single stranded nucleic acids, double-strandednucleic acids, partially double-stranded nucleic acids, and/orcombinations thereof. In one embodiment where the nucleic acids aresingle stranded nucleic acids, one or more complementary nucleic acidoligomers are hybridized with the single stranded nucleic acids in alength or under conditions that provide for sufficient stability tomaintain at least a partially double-stranded form for testing. In oneembodiment where the nucleic acids include a strand of RNA forsequencing, by using the disclosed method, no reverse transcriptase oroptical detection method is used for sequencing the strand of RNA. Inone embodiment where the nucleic acids include double stranded DNA, byusing the disclosed method, the double stranded DNA is sequenced withoutmaking the double stranded DNA single-stranded. In embodiments, thedisclosed method may be a signal detection method using achloride/silver/silver chloride measuring system.

In accordance with various embodiments, there is provided a method formolecular separation, purification, and sensing. In such a method, oneor more nanowells or nanotubes or combinations thereof may be providedin a support material with each nanotube including one or more sidewallelectrodes and/or each nanowell including one or more sidewallelectrodes and/or one or more bottom electrodes. One or moreelectroactive nanomembranes may also be provided on at least a portionof the one or more sidewall or bottom electrodes or combinations thereofto form a nanopore or a nanochannel by the one or more electroactivenanomembranes. The one or more electroactive nanomembranes may befunctionalized by attaching nucleases thereto. Nucleic acids may beplaced above the nanopore or nanochannel, wherein nucleic acid bases areformed from the nucleic acids by the enzymatic activity of thenucleases. The nucleic acid bases may then be moved towards and throughthe nanopore or the nanochannel by applying an ion funnel current orvoltage. A sequence of the nucleic acids may then be derived bycomparing measured signals of a passage of the nucleic acid basesthrough the nanopore or the nanochannel with predetermined signals. Inone embodiment where the nucleic acids include a strand of RNA forsequencing, by using the disclosed method, no reverse transcriptase oroptical detection method is used for sequencing the strand of RNA. Inone embodiment where the nucleic acids include double stranded DNA, byusing the disclosed method, the double stranded DNA is sequenced withoutmaking the double stranded DNA single-stranded. In embodiments, thedisclosed method may be a signal detection method using achloride/silver/silver chloride measuring system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a portion of an exemplary device including ananowell array according to various embodiments of the presentteachings.

FIGS. 2A-2C are schematics showing nanomembrane deposition using one ormore materials and tunability on sidewall of an exemplary nanowellaccording to various embodiments of the present teachings.

FIGS. 3A-3D depict analyte motion-inducing methods using devices ofFIGS. 1 and/or 2A through 2C according to various embodiments of thepresent teachings.

FIGS. 4A-4B depict exemplary embodiments of DNA sequencing.

FIGS. 5A-5B depict exemplary embodiments in monitoring DNAhybridization.

FIGS. 6A-6B depict exemplary embodiments in monitoring antigens bindingto antibodies.

DETAILED DESCRIPTION

The present disclosure describes an apparatus and methods for molecularmanipulation system that is sufficiently flexible that it can beemployed to separate and purify molecules as structurally and chemicallydiverse as proteins, nucleic acids, ions, and small molecules. The sameunderlying technology can be combined with sensors such as optical andelectrical detectors to identify and quantify this same breadth ofmolecules. Further specializations of the underlying technologies openentirely new applications, too, for example including a highly scalableand robust system for sequencing DNA and RNA molecules. The keyinnovation enabling such a broad spectrum of capabilities relies on anovel apparatus and methods by which nanostructures, in particularnanomembranes, can be manufactured such that they can provide fortunable dimensioning. These tunable nanomembranes furthermore can beeasily engineered to select for their bulk and surface properties,and/or functionalization, to enable the manipulation and quantificationof a very wide range of molecule types.

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. In embodiments, terms such as “an array” or “the nanowell”may include a plurality of arrays or nanowells, respectively, unless thecontext clearly dictates otherwise.

In embodiments, the terms “up” and “down” connote only that theyrepresent a general directional flow of a given material and that, whenused in the same context, represent generally opposing flowdirectionalities.

In embodiments, the term “bulk,” as in the “bulk” of a nanomembrane or ananomembrane's “bulk” properties, do not necessarily connote anyabsolute proportionality as to how much of the entirety of a givenproperty is due to the “bulk” versus the remainder that differs from the“bulk.” As a non-limiting example, a thin film of one nanomembranematerial can be layered over the bulk of another membrane material suchthat the thin film occupies 5-10% of the nanomembrane's total volume.This thin film may be induced to occupy 40-50% of the samenanomembrane's total volume when it is subjected to givenelectroactivity-inducing imposed electrical stimuli. Thus, “bulk” versusnot “bulk” is necessarily contextual in the provided embodiments.

“Solid support,” “support,” and “support material” are referred toherein as a material or group of materials having a rigid or semi-rigidsurface or surfaces. In some aspects, at least one surface of the solidsupport will be substantially flat, although in some aspects it may bedesirable to make use of solid supports bearing, for example, raisedregions, pins, etched trenches, and/or the like. A variety of materialscan be used for the solid support including, but without limitation,silicon/silicon derivatives, polymers, ceramics, glass, metals, and/orother possible materials. Multiple materials may be used to manufacturea given solid support.

A solid support may be manipulated such that the mode(s) by which itsupports a given structure or structures can be altered during thecourse of its manufacture and use. In embodiments, the solid support maybe subjected to manipulations such as chemical or plasma etching tochange the three-dimensional manner in which it supports thesestructures. The solid support also may be derivatized to change thecomposition of the material in which the support of the structures isprovided. In embodiments, a solid support can be derivatized with one ormore molecules known to modify its surface properties to make it morehydrophobic, hydrophilic, ion impermeable, chemically reactive, and/orelectrically insulating. Multiple cycles of solid support derivatizationwith the same or different molecules also can be employed. The solidsupport also can be coated with one or more materials. Such surfacecoatings also can allow a desired solid support in embodiments to makeit more hydrophobic, hydrophilic, ion impermeable, chemically reactive,and/or electrically insulating. These coatings can be added or removedrepeatedly during the manufacture and use of a given solid support.

A “chamber” is a space that occurs over the surface of a solid support.This space may be defined by some type of package or cell thateffectively forms an enclosure over a solid support's surface. A chambermay be formed with essentially any dimensionality, and may also includeone or more inlets and outlets for example to allow for fluids and/orgases to be passed over the solid support's surface. More than onechamber may be placed over the solid support's surface, too. Chambersmay be manufactured such that they can be readily placed or removed,materials can be transferred or not between them, etc. These chamberconfigurations also can be modified any number of times over the courseof a given application. Chambers can be formed of any material that iscompatible with a given application, for example including, but withoutlimitation, silicon/silicon derivatives, polymers, plastics, rubber,ceramics, glass, metals, and/or other possible materials. Chambermaterials also can be changed over the course of the manufacture and useof a given solid support, for example, but without limitation, makinguse of the same or different derivatizations and coatings used to modifya solid support.

An “array” is an intentionally created collection of two or morestructural elements on, and/or in, a solid support. Arrays aremanufactured by wide variety of means, for example making use of thediverse methods of the semiconductor manufacturing industry. Thus,arrays could take a wide variety of forms, for example bearing two ormore electrodes, nanowells, nanotubes, and/or trenches, and so forth. Anarray could either be a macroarray or a microarray, depending on thesize of the structural elements on the solid support. A macroarraygenerally contains structural element sizes of about 300 microns orlarger. A microarray would generally contain structural element sizes ofless than 300 microns. However, it should be noted that macroarrays andmicroarrays share essentially similar properties, differing in the artfor example regarding their dimensional scales, how they aremanufactured, and/or how materials are deposited upon them. Thus, theterm “array” is used herein to describe both macroarrays and microarraysfor the invention herein, and it should be understood that this term isnot intended to actually denote any dimensionality specifications orlimitations. The total surface area of an array can significantly exceedthe scale of the structural elements on its surface; microarrays forexample may occupy multiple square centimeters of a solid support andmacroarrays may occupy less than a square centimeter of solid support.

“Functionalized arrays” are arrays that have been further modified withmolecules. These molecules may be prepared synthetically,biosynthetically, and/or purified, and then attached to an array tofunctionalize it. Functionalization may also be done “in situ” such thatmolecules are synthesized, purified, etc in place on an array's surface,for example, but not limited to, making use at least initially of one ormore of the electrochemical reactions described herein. The moleculesused to functionalize an array can be identical or different from eachother on a given array. Furthermore, functionalized arrays can assume avariety of formats, in embodiments, bearing polymers, libraries ofsoluble molecules; and/or libraries of molecules tethered to polymers.The molecule libraries used for functionalization can include, but arenot limited to, proteins, enzymes, antibodies, antibody fragments,phage, nucleic acids (DNAs, RNAs), aptamers, peptides, ions, smallmolecules, drugs, and/or combinations thereof. Exemplary enzymes caninclude kinases, phosphatases, nucleases (DNA and RNA cleaving),synthases (e.g., DNA polymerases), oxidases (e.g., glucose oxidases),peroxidases, reductases, and/or their combinations. Suitable chemistriesfor the functionalization can include the use of cross-linkers,heterobifunctional cross-linkers, and/or light-activated cross-linkers.Other chemistries, such as electrochemical coupling reactions throughone or more corresponding electrodes, can also be used.

A “well” or a “nanowell” is a three-dimensional structural element thatis manufactured to form a depression into a solid support. The overalldimensions of a well or nanowell can be varied tremendously dependingupon the manufacturing methods and the nature of the solid support used.A “well” describes this structural element with its smallest dimension(e.g., diameter, depth) larger than about 300 microns. A “nanowell”describes this structural element with its smallest dimension smallerthan about 300 microns. However, it should be noted that wells andnanowells share essentially similar properties, differing in the art forexample regarding their dimensional scales, how they are manufactured,and/or how materials are deposited upon them. Thus, the term “nanowell”is used to describe both wells and nanowells for the invention herein,and it should be understood that this term is not intended to actuallydenote any dimensionality specifications or limitations. Similarly, anarray of two or more actual nanowells and/or wells is summarized byreference herein as being an array of nanowells even though it should beunderstood that both wells and nanowells can be manufactured in the samesolid support. Although the nanowells shown in figures herein have anessentially circular cross section, this dimensionality is actually usedfor illustrative purpose. One of ordinary skill in the art willappreciate that any other, regular or irregular, dimensionalities ofnanowells are contemplated to accommodate various embodiments of thepresent teachings. In fact, nanowells can have essentially anydimensionality or (a)symmetry, and may be limited, for example, only bythe capabilities of the manufacturing method used to form a given solidsupport, or the application for which it is being made. Thus, nanowellscan have a dimensionality in their cross-sections including, but notlimited to, circular, square, rectangle, polygon, or any other suitablecross-section dimensionalities, and they also can have essentially nodiscernable shape in their cross-sections. A nanowell can have straightsidewall. Alternatively, a nanowell can have non-straight sidewalls, forexample, having one or more overhangs, indentations, lips, steps,cupping, bulges, angles, etc., and these features can differ anywherealong the sidewall of the nanowell. A given solid support can bemanufactured to have one nanowell, or to have one or more arrays ofnanowells, and these multiple nanowells can have the same or differentdimensionalities and (a)symmetries.

As defined herein, a cross-section of a nanowell relates to atwo-dimensional representational view of a given example of thisstructure that largely bisects across its sidewall. A lateral-section ofa nanowell relates to a two dimensional representational view of a givenexample of this structure that that largely runs in parallel with itssidewalls. For a nanowell with varying/irregular sidewall profiles,however, cross-section(s) and lateral section(s) representations maysignificantly overlap.

Various configurations of nanowells in an array can be used. Inembodiments, a nanowell array can be configured periodically ornon-periodically. The nanowells in an array can be aligned in square,hexagonal, or any other pattern, or can lack any alignment or pattern.Nanowell arrays also can have nanowells placed in one or more patternson one portion of their surfaces, while other portions have nanowellsplaced in no particular pattern.

“Tubes” and “nanotubes” are depressions formed in a solid support suchthat they effectively lack a bottom, or at least that they lack a bottomthat is directly attached to the sidewall as with a nanowell. Thus, atube or nanotube entirely pierces a given solid support. The overalldimensions of tubes and nanotubes can be varied tremendously dependingupon the manufacturing methods and the nature of the solid support used.A “tube” describes this structural element with its smallest dimensionlarger than about 300 microns. A “nanotube” is a structural element withits smallest dimension smaller than about 300 microns. However, itshould be noted that tubes and nanotubes share essentially similarproperties, differing in the art for example regarding their dimensionalscales, how they are manufactured, and/or how materials are depositedupon them. Thus, the term “nanotube” is used to describe both tubes andnanotubes for the invention herein, and it should be understood thatthis term is not intended to actually denote any dimensionalityspecifications or limitations. Similarly, an array of two or more actualnanotubes and/or tubes is summarized by reference herein as being anarray of nanotubes even though it should be understood that both tubesand nanotubes can be manufactured in the same solid support.

As used herein, a cross-section of a nanotube relates to atwo-dimensional representational view of a given example of thisstructure that largely bisects across its sidewall. A lateral-section ofa nanotube relates to a two dimensional representational view of a givenexample of this structure that largely runs in parallel with itssidewalls. For a nanowell with varying/irregular sidewall profiles,however, cross-section(s) and lateral section(s) representations maysignificantly overlap.

Nanotubes may be formed essentially in any dimensionality and, as withnanowells, and their “depth” is only limited by the thickness of thesolid support in which they are formed. Although the nanotubes shown infigures herein have a circular cross section, this dimensionality isactually used for illustrative purpose. One of ordinary skill in the artwill know that any other, regular or irregular, dimensionalities ofnanotubes are contemplated to accommodate various embodiments of thepresent teachings, as with nanowells. When more than one nanotube isformed in a solid support, the resulting structure becomes a nanotubearray. The number of nanotubes formed, their arrangement with respect toeach other, and that each may be identical or in any combination(s) ofdistinctiveness with respect to each other is possible in embodiments.One or more nanotubes may form a connection between a single chamber,between two distinct chambers, between any number of distinct chambers,and/or between any combinations of connected or disconnected chambers.

Nanotubes also can be made to form a connection between two othernanotubes in a given solid support. The dimensionalities of such“connecting nanotubes” formed between two or more nanotubes within asolid support can be as varied as that of the nanotubes themselves.Thus, since connecting nanotubes can have essentially the sameproperties as nanotubes, connecting nanotubes also are referred toherein as “nanotubes.”

It is well known in the manufacturing industry that two surfaces can becombined together in a precise joined orientation and then appropriatelyfixed together for example with clamps, glues, solder, and the like. Inthis manner, three-dimensional structural elements can be formed by thecombination of two or more solid support as the surfaces are broughttogether. Thus, these methods can be used for example to form nanotubesand/or nanowells, as well as arrays thereof, which result from thecombination of two or more solid supports.

A “nanochannel” is defined herein as the open space or void within asolid support that is bounded by a given nanotube's sidewall andsidewall electrodes, as well as the void within a solid support that isbounded by a given nanowell's sidewall and bottom, and sidewall andbottom electrodes. As such, the dimensionalities of a given nanochannelcan be as varied as the wide range of dimensionalities of the possiblenanotubes and/or nanowell structures described herein. Similarly, thetwo or more nanochannels in a given solid support can have the sameand/or markedly different dimensionalities just as the nanotube(s)and/or nanowell(s) bounding the nanochannels can have the same and/ormarkedly different dimensionalities in that solid support. Nanochannelsalso can be formed by the combination of two or more solid supports justas nanotubes and/or nanowells can be formed in the same manner.

As defined herein, a cross-section of a nanochannel relates to atwo-dimensional representational view of a given example of thisstructure which largely bisects across its outer perimeter as providedby a given combination of dimensionalities of a nanowell's or nanotube'ssidewall, sidewall electrodes, bottom, and nanomembranes. Alateral-section of a nanochannel relates to a two dimensionalrepresentational view of a given example of this structure which largelyruns in parallel with its outer perimeter. For a nanochannel withvarying/irregular perimeter profiles, however, cross-section(s) andlateral section(s) representations may significantly overlap.

As used herein, an “electrode” is an electrically active surface thatcan be made of any suitable conductive material or materials. When morethan one electrode is placed on a solid support, as in an array, thesecan be the same or different conductive materials. The conductiveelectrode material can for example, but is not limited to, metals,conductive polymers, carbon-based conductive structures, conductivecomposites, and/or combinations thereof. Exemplary metals can includesilver, gold, platinum, copper, titanium, aluminum, tungsten, and/ortheir combinations. Exemplary conductive polymers can be polyacetylene,polyphenylene vinylene, polypyrrole, polythiophene, polyaniline,polyphenylene sulfide, and/or their combinations. Exemplary carbon-basedconductive structures can include carbon films, plates, tubes, rods,buckyballs, graphene, and/or their combinations. Exemplary conductivecomposites include electrically conductive metal matrixes, epoxies,adhesives, silicones, laminates, elastomers, carbonized plastics, and/ortheir combinations.

Optionally, electrodes can be made of one or more materials that arefurther useful in providing for additional functionalities beyondelectrical conductivity. In embodiments, metals can be used to form anelectrode which can undergo a chemical reaction in the presence of anappropriate molecule such that they are become oxidized or reduced. Suchan oxidation or reduction can result in the electrode becomingpositively or negatively charged, a result that can be monitored by awide range of methods known in the art. Silver metal undergoes thesilver/silver chloride reaction in the presence of chloride ions is oneexample of many known in the art for how such a functionality can bemade available for nanotube and nanowell electrodes.

Electrodes also can be subjected to one or more derivatizations toimpart other functionalities in addition to conductivity. Inembodiments, metals such as silver and platinum can be reacted withoxygen to form an oxide. Such a metal oxide may be further reacted, forexample, with the deposited nanomembrane material described herein suchthat the two materials become covalently bonded together. Thus, bychoosing which electrodes are derivatized or not, this can serve as amethod for selecting which electrode becomes a deposition site or not.

Electrodes also can be coated with one or more materials to provide forfunctionality in addition to conductivity. In embodiments, an electrodecan be coated with materials to provide for chemical reactionresistance, electrical passivation, a hydrophilic or hydrophobicsurface, a neutrally charged surface, a positively or negatively chargedsurface, etc. Such electrode coatings also can allow a desired electrodeor a group of electrodes to be used as reaction sites forelectrochemical synthesis and/or for inhibiting correspondingelectrode(s) from participating and/or being affected by any possiblechemical reactions in embodiments.

It should be noted, though, that a wide range of other additionalfunctionalities, derivatization methods, and coating methods are wellknown in the art for the conductive electrode materials describedherein, and these also can be used to impart a similarly wide range ofcapabilities beyond the examples described. Such functionalities,derivatives, and coatings can be deposited and removed repeatedly duringmanufacturing and use of the apparatus provided herein.

Each nanowell and/or nanotube can include one or more electrodesconfigured along its sidewall, which are referred herein as “sidewallelectrode(s).” Furthermore, a nanowell may have one or more electrodesconfigured at the bottom of the nanowell, which are referred herein as“bottom electrode(s).” In embodiments, one or more “bias electrodes”also can be included. A bias electrode is an electrode that is locatedelsewhere other than the sidewall or bottom of the one or more nanowellsand/or nanotubes in a solid support. Thus, a bias electrode or biaselectrodes may be placed on the solid support's surface(s) (i.e., onareas of a given solid support that is not part of a nanotube ornanowell), and/or placed in the one or more chamber onto which the solidsupport is attached or mounted. Note that in the range of possibledimensionalities of nanotubes and nanowells that it can be ambiguous asto whether a given electrode is a sidewall, bottom, or bias electrode.It also can be useful to manufacture electrodes that cross multiplesurfaces of nanotube or nanowell (e.g., spanning across the bottom,sidewall, and out onto the surface of a nanowell). Nonetheless, thesetypes of electrodes can have the same general dimensional and electronicproperties as the more obviously defined sidewall, bottom, and biaselectrodes. Therefore, these types of electrodes are referred to hereinas sidewall electrodes, even though their particular location(s) may notbe clearly limited to the confines of a given sidewall.

Sidewall electrodes can be configured in any dimensionality, so long asthey remain at least partially placed on the sidewall of a givennanowell or nanotube. For example, sidewall electrodes can bemanufactured such that they partially to completely encircle/ring ananotube or nanowell's sidewall. Sidewall electrodes also can be craftedto have essentially any dimensionality, for example being rectangular,circular, banded, asymmetric, etc, and each can be independentlyoperable electronically. Sidewall electrodes also can be formed bybreaking an electronically connected electrode into two or more segmentsalong the sidewall, wherein the broken segments can have same ordifferent dimensionalities that are separated by electrically resistivematerials. Such electrically resistive material can be, but is notlimited to, silicon dioxide, silicon nitride, polyimide(s), photoresistmaterials, and combinations thereof. Any distance can separate sidewallelectrodes from one another so long as they remain associated within theconfines of a given sidewall. These segmented electrodes may beconnected to the same or independant electronics elements (inembodiments, capacitors, voltage sources, current sources, resistors,amplifiers, analog-to-digital converters, sensors, etc). Sidewallelectrodes also can be placed in any configuration such that they occuras stacks of electrodes separated by electrically insulatingmaterial(s). Theses stacked electrodes, too, may be connected to thesame or independent electronics elements. The types of sidewallelectrodes employed in a nanotube or nanowell array may be the same ordifferent, being limited only by the mode of manufacturing used to formthem, the application for which they are intended, and the type ofelectrical controls used to operate them.

Bottom electrodes of a nanowell have a similar flexibility indimensionality as that of the sidewall electrodes so long as they remainat least partially associated in the confines of the bottom of ananowell. Bottom electrodes may be any dimensionality, for example beingsquare, circular, ringed, asymmetric, etc. In the instances in whichmore than one bottom electrode is placed in a nanowell, these also canbe placed to be in no particular pattern with respect to each other,and/or they may be patterned such as in a grid, as concentric elements,etc. When one or more bottom electrodes are utilized, these can beseparated from each other by some electrically insulating material suchas, but not limited to, silicon dioxide, silicon nitride, polyimide(s),photoresist materials, and combinations thereof. Bottom electrodes canbe separated from one another by any distance so long as they remainassociated within confines of a given well's or nanowell's bottom.Bottom electrodes, too, may be connected to the same or independentlyoperating electrical elements.

Bias electrodes have the same flexibility in their dimensionalities andplacement with respect to each other as is provided for the otherelectrode types described herein.

The sidewall, bottom, and bias electrodes in the one or more nanotubesand/or nanowells in a solid support need not be made to be flush withthe solid support surface. In embodiments, these electrodes can bemanufactured such that they are placed in depressions within and/orextensions above the local surface of the solid support or chamber.Similarly, these electrodes themselves can form a depression withinand/or extension above the local surface of the solid support orchamber. These electrodes also can be manufactured such that theygenerally follow the plane of the local solid support surface at oneplace and yet are within depressions below and/or extensions above thelocal solid support in another place(s). Similarly, the electrodematerial itself can be fashioned to further define the topology of alocal surface, for example by being manufactured to have a depression(s)and/or extension(s) that may or may not reflect the contours of thelocal solid support surface.

In the case of nanotubes, one or more secondary and/or bias electrodescan be employed to serve as the pseudo-bottom electrode(s).Pseudo-bottom electrodes can be the sidewall and/or bottom electrode(s)in the nanowell(s) facing away from the ‘top’ chamber of a nanotube.They also can be the bias electrode(s) on the surfaces of either or bothsides of a solid support containing a nanotube, and/or in the chamber(s)attached to either or both sides the solid support. Pseudo-biaselectrodes have the same flexibility in their dimensionalities andplacement with respect to each other as is provided for the otherelectrode types described herein.

During operation, the sidewall electrodes and/or/or the bottomelectrodes and/or the bias electrodes each can be operatedindependently, in series, and/or in parallel to accommodate a givenrequirement. These operational modes furthermore can be changed overtime as required. Additional circuitry providing for all electronicoperations as known in the art also may be incorporated in the solidsupport and connected to the electrodes. In embodiments, the electrodescan be connected to electrical components found in the art including,but not limited to, components that provide electrical connectivity byany appropriate mode known in the art; control and delivery of voltage,current, and/or resistance, including from being invariant to enablingany desired variation in amplitude, frequency, and/or wave form by anyappropriate mode known in the art; signal sensing, amplification, noisereduction, smoothing, analog-to-digital conversion, and/or any othersignal processing by any appropriate mode known in the art; datacommunication in analog or digital formats by any appropriate mode knownin the art; data storage in analog or digital formats by any appropriatemode known in the art; and/or any combinations of the above.

In instances in which two solid surfaces are combined together to formnanotubes and/or nanowells, it is well known in the industry thatelectrical circuitry can be connected across the resulting junction ofthe two solid supports if this is desired. In the semiconductorindustry, for example, one mode for making such electrical connectionsis to employ a technique called making a “flip-chip” or a “controlledcollapse chip connection.”. In this manner, three-dimensional electricalelements can be formed as the surfaces are brought together. Thus, suchmethods can be used for example to form one or more electrodes thatconnect across tubes, nanotubes, wells, and nanowells that are formed bythe combination of more than one solid supports. Such electricalelements like electrodes can have all of the same dependent and/orindependent functionalities of the bottom, sidewall, and biaselectrode(s) described for nanowells and/or nanotubes in a single solidsupport. It should be noted that the particular mode by which flip-chipsare generally formed in the silicon industry does not limit the means bywhich the apparatus described herein can be manufactured. Two or moresolid supports also can be brought together in an acceptably stable andfunctional complex via the use of glues, thermal fusion, etc., as isappropriate for a given application. It may be desirable to bring two ormore solid support surfaces for reasons other than forming newelectrical connections, though. For example, surfaces may be broughttogether simply to form other topologies that make no furthermodification(s) to their pre-existing electrode configurations. In anycase, whether, as defined herein to indicate the formation of newstructures by bringing together one or more solid support for whateverreason and/or by whatever method, the resulting apparatus is referred toherein as being a “flip-chip.”

As used herein, the term “membrane” or “nanomembrane” refers to anelectroactive material or layer of materials that is deposited on one ormore electrodes. As used herein, the electrode upon which a membrane ornanomembrane is deposited is a “primary electrode.” Primary electrodescan be one or more sidewall, bottom, or bias electrode, or anycombinations of these electrodes. These primary electrodes can beelectronically interconnected and/or independently operable. A“membrane” generally refers to a layer of electroactive material that isdeposited over the larger sized electrode structures that generally areassociated with tubes and wells, while a “nanomembrane” is depositedover smaller sized electrode structures such as those generallyassociated with nanotubes and nanowells. However, it is understood thatthe properties of membranes and nanomembranes essentially can be thesame except for how these differ due to their lesser or greaterthickness, distances covered, etc. Thus, the term “nanomembrane” is usedto describe both membranes and nanomembranes herein, and it should beunderstood that this term is not intended to actually denote anydimensionality specifications or limitations. Similarly, in the instancein which a nanotube and/or nanowell array bearing two or more actualnanomembranes and/or membranes, such a system is summarized by referenceherein as being an array bearing nanomembranes even though it should beunderstood that both scales of these structures can be manufactured inthe same solid support.

It can be desirable to deposit an “electroactive nanomembrane” on asidewall primary electrode that encircles the interior of a nanotubeand/or a nanowell. These encircling primary sidewall electrodes can bemanufactured either directly in the a sidewalls of a nanotube and/ornanowell, or they can be formed as two or more solid supports containingportions of these structures are brought together as per the “flip-chip”methodologies as described herein. In either case, the resultingdeposited nanomembrane on such encircling electrodes can form ring ofdeposited material that also encircles the interior of a nanotube and/ora nanowell. As defined herein, this apparatus and methodology results inthe formation of an “encircling nanomembrane.” Illustrations providedherein of encircling nanomembranes are depicted as being an annulus(i.e., being essentially circular or donut-shaped in its profile).However, encircling membranes in fact can be deposited in any shape. Forexample, when deposited on cylindrically shaped surfaces, a thinlydeposited encircling nanomembrane will generally conform to thiscylindrical shape. A more thickly deposited encircling nanomembranenonetheless can assume any shape that may or may not be essentiallycircular. Note that one or more encircling nanomembranes can bedeposited on the interior of a given nanotube or nanowell, as more thanone encircling electrode can be placed with the solid supports of thesestructures.

As used herein, unless otherwise specified, the term “nanomembrane”generally refers to “electroactive nanomembrane” in this disclosure,although one or more non-electroactive nanomembrane(s) can be used incombination with the electroactive nanomembrane(s). In embodiments,electroactive nanomembrane(s) can be formed over nanomembrane(s) thatare electroactive and/or non-electroactive on corresponding electrode(s)as disclosed herein. In embodiments, non-electroactive nanomembrane(s)also can be formed over nanomembrane(s) that are electroactive and/ornon-electroactive on corresponding electrode(s) as disclosed herein.

In any case, the presence of a given encircling nanomembrane contributesto the dimensionality of the nanochannel as described herein, and thiscontribution can change in response to the given nanomembrane'selectroactivity in response to applied electrical stimuli.

Illustrations of the interior cross-section of an encirclingnanomembrane are depicted herein as defining a space that is essentiallycircular. However, the interior cross section of the nanochannel that issurrounded by an encircling nanomembrane can be essentially any shape.Thus, regardless of its interior cross-section shape, the nanochannelthat is surrounded by an encircling nanomembrane is defined herein asbeing a “nanopore.” A nanopore can be of any dimension along itscross-section and lateral-section so long as it remains surrounded by anencircling nanomembrane. In addition, while it can be desirable for ananopore to be manufactured such that it essentially is centrally placedin a nanowell or nanotube, this is not a requirement (in embodiments,one or more nanopores can be placed anywhere in an encirclingnanomembrane). Note that one or more nanopores can be formed in anencircling nanomembrane with a relatively thin lateral-section. In suchinstances, the lateral-section of such a nanopore is comparatively small(in embodiments, but not limited to, tens to hundreds of nanometers inlateral-section). Similarly, one or more nanopores can be formed inencircling nanomembranes of very long lateral-section in embodiments,but not limited to, forming one or more nanopores in an encirclingnanomembrane spanning more than a millimeter in its lateral-section). Agiven nanopore's lateral-section and cross-section also may vary alongthe length of the nanochannel that forms it, so long as it remainsentirely encircled by nanomembrane.

The term “electroactive” refers to a property of a material thatexhibits electrical activity or response to electrical stimuli. Thedeposited nanomembrane material or layer of materials described hereincan be electroactive (i.e., having “electroactivity”) in that itundergoes a dimensionality change in response to imposed electricalstimuli such as electrical current, voltage, or electrical waveformprovided at the one or more primary electrodes upon which it isdeposited. However, the deposited nanomembrane material or layer ofmaterials could be non-electroactive nanomembranes in the system. Notethat nanomembrane electroactivity also can be elicited by imposingelectrical stimuli at one or more electrodes other than the primaryelectrode, herein described as “secondary electrodes.” Such secondaryelectrodes can be one or more sidewall, bottom, and/or bias electrodes.Furthermore, the electrical stimuli can be provided by combinations ofprimary and/or secondary electrodes.

As used herein, a “dimensionality change” describes any modification ofa nanomembrane's three-dimensional conformation resulting from imposedelectrical stimuli at one or more primary or secondary electrodes. Adimensionality change can include, but is not limited to, an increaseand/or a decrease in a nanomembrane's length, thickness, breadth,bending, twisting, looping, etc. Such dimensionality changes may or maynot be accompanied by a change in a nanomembrane's density. Suchdimensionality changes also may or may not be accompanied by a change inthe nanomembrane's molecular composition, and/or or chemical bondstructure. A nanomembrane's dimensionality change may or may not includethe addition or removal of non-covalently bound or covalently boundmaterial from its surface, or within its internal structure. Thus, forexample, a dimensional change may occur via the polymerization ordepolymerization of just one molecule at the surface of a pre-existingnanomembrane's surface or within its interior. Therefore, inembodiments, the scale of a nanomembrane's dimensional change asdescribed herein can proceed via the formation or breaking of a singlechemical bond, and/or via a compositional change as small as theinclusion or removal of a single molecule in non-covalent and/orcovalent association with a given nanomembrane.

As used herein, making use of a nanomembrane's “electroactivity” entailsemploying instrumentation and methods to control the imposition ofelectrical stimuli such as, but not limited to, a voltage or current ina given electrical waveform to an electrode of interest in order toinduce a nanomembrane's dimensional change. There are a wide variety ofvoltage, current, and electrical waveform control apparatus and methodswell known in the art to provide for this use. These include, but arenot limited to, apparatus and methods providing for the generation of: adirect current; an alternating current; a current or voltage modulatedas a sine, square, saw-tooth, etc. waveform; a cyclic voltammetry-drivencurrent or voltage; an electric waveform supplied at constant voltage,constant current, or constant power; voltages and/or currents varying inany type of amplitude and/or frequency; voltages a; and/or currentsvarying in their duration and/or number of pulses; and combinationsthereof. While it is understood that the instrumentation and methods toenable controls for imposing a wide variety of apparatus and methods forimposing voltages, currents, and/or electrical waveforms at an electrodeare known in the art, the breadth of these available options aresummarized by reference herein as imposing “electrical stimuli” at anelectrode.

Thus, in the instance in which one or more nanomembranes is deposited ona sidewall and/or bottom of a nanowell, and/or the sidewall of ananotube, an electroactive nanomembrane's, or nanomembranes',contribution to the dimensionality of the nanochannel(s) can change asit is subjected to the imposition of electrical stimuli.

Nanomembranes can be deposited by a wide range of manufacturingprocedures. Non-electrochemical methods include, but are not limited to,methods such as spraying, vapor-phase deposition, sputtering, spincoating, precipitation, in situ polymerization by means such as heat-and/or photo-curing, multilayer deposition with the well layer being asacrificial material that is etched away to create a material membraneover a well, etc.

Such a deposited membrane can be in close, but non-covalently attachedproximity, to the given primary electrode, or it can be directlyattached to the given primary electrode such as via covalent chemicalbonding. The latter instance generally requires the electrode to becomposed of a material that can undergo chemical bonding, or it has beenderivatized and/or coated with a material that enables chemical bondformation to occur.

Examples of electroactive nanomembrane materials that can be depositedon primary electrodes include, but are not limited to, dielectricelectroactive polymers in embodiments (e.g., certain types of siliconeand acrylic elastomers), ferroelectric electroactive polymers inembodiments (e.g., polyvinylidene fluoride polymers), ionicelectroactive polymers in embodiments (e.g., certain conductivepolymers, ionic polymer-metal composites, and responsive gels), and/ortheir combinations.

Some deposited electroactive nanomembrane materials can be induced tochange dimensionality even during their deposition in response to theimposition of electrical stimuli at the primary electrode(s) upon whichthey are being deposited. Some electroactive nanomembrane materials alsocan be induced to change dimensionality during their formation viaelectrical stimuli being imposed at the secondary electrode(s) in thesolid support and/or the chamber(s).

Nanomembranes also can be electroactive in that these membranes aredeposited in an electrically tunable dimensionality at the primaryelectrode(s) by imposing electrical stimuli that induces chemical bondformation of monomers into polymers via direct and/or indirectelectrochemical reactions. Thus, such electroactive nanomembranes aredeposited by electrochemical means. Electrochemical reactions of certainmonomers to form such electrically conductive polymers such asnanomembranes are well known in the art (see Malinauskas et al.,Nanotechnology, 2005). Thus, as sufficient electrical stimuli areimposed to the desired primary electrode(s), certain electroactivemonomer molecules in a solution contacting that electrode can be inducedto undergo chemical bond formation that results in a nanomembranedeposition. The resulting nanomembrane either can be in close, butnon-covalently bonded, proximity to one or more primary electrodes, orit can be covalently attached to one or more primary electrodes. Asdescribed herein, an electrochemically deposited nanomembrane is atleast initially electrically conductive such that imposed electricalstimuli at the primary electrode(s) upon which the nanomembrane is beingdeposited can propagate through the nanomembrane itself. Therefore,imposed electrical stimuli at a primary electrode can transfer throughthe electrically conductive nanomembrane such that additional monomer atleast initially is incorporated into the nanomembrane elsewhere alongits surface, and/or polymer is removed from its surface. For thisreason, the dimensionality of the electrochemical reaction to form suchnanomembranes thereby is electrically tunable in its dimensionalitybased upon whether or not electrical stimuli are applied at the primaryelectrode(s).

The one or more monomers chosen for example in a later stage ofelectrochemically-induced deposition also can be selected for propertiessuch that the resulting polymer material has diminished to entirelylooses electrical conductivity. Such types of monomers, therefore, canbe chosen to effectively terminate an electrochemical depositionreaction at a given primary electrode(s). Such a process can be usefulto diminish or eliminate the dimensional tenability of one or moreelectroactive nanomembranes, while preserving electrical tenability atother electroactive nanomembranes.

Some electrochemically deposited nanomembranes also can be chosen to becomposed of materials that can be induced to change dimensionalityduring a desired deposition reaction that also responds to an impositionof electrical stimuli at the secondary electrode(s). In such instances,one or more secondary electrode can be energized with electrical stimulithat are too low to provide for polymer formation (i.e., so that they donot themselves become primary electrodes upon which polymer nanomembraneformation occurs), but with sufficient electrical stimuli to contributeto the dimensionality/conformation of the nanomembrane being formed on aprimary electrode(s). Similarly, the secondary electrodes also can bederivatized or coated such that they themselves are not subjected to anelectrochemical deposition processes despite being subjected to imposedelectrical stimuli that otherwise could induce nanomembrane formation.

Note that some deposited electroactive membranes, whether deposited byelectrochemical or non-electrochemical means, also can be depolymerizedor have their deposited material removed by both non-electrochemicaland/or electrochemical means. For example, nanomembranes can bedeposited using energetic neutral atom beam lithography/epitaxy asdescribed in U.S. Pat. No. 7,638,034.

Non-electrochemical and/or electrochemically deposited nanomembranes canbe chosen to be composed of materials with rigidity such that theymaintain a desired dimensionality only while one or moredimensionality-modulating electrodes are no longer exerting theirimposed electrical stimuli-driven force effects on the polymer.Furthermore, nanomembranes also can be composed of materials withrigidity such that its dimensionality modulation is maintained, or evenfurther modulated after its manufacture while under imposed electricalstimuli-driven force effects.

Electrically tunable nanomembranes, whether deposited electrochemicallyor non-electrochemically by other means, can be deposited virtually inany dimensionality. The embodiments of their dimensionalities arelimited only by matters such as the availability of manufacturingmethods, the stability of the nanomembrane post-manufacturing,scalability, achieving the properties required for a given application,etc.

In the instances in which a given deposition method results in amolecular monolayer or similarly thin nanomembrane at the primaryelectrode(s), the deposited nanomembrane's dimensionality generallyconforms to the dimensionality of the primary electrode(s) itself.However, it also can be desirable to employ deposition methods to form agiven nanomembrane that project significantly into a nanowell's ornanotube's interior. More deeply deposited nanomembranes can be in avery wide variety of forms including, but not limited to, formingstructures such as rods, loops, wedges, fans, layers, tubes, cones,interwoven lines, sheets, etc.

Electrochemical and other deposition methods can provide fornanomembranes that join two or more electrodes. In the instances inwhich the deposited material is electrically conductive, the resultingnanomembrane connecting two or more electrodes can function essentiallyas an electrical connection between these electrodes.

In the instance of an electrochemical polymerization method being usedto drive nanomembrane deposition, a sidewall primary electrode can beemployed as the initial site of polymer deposition and then one or moresecondary electrodes can be appropriately energized to attract thegrowing polymer towards their surface. Such a reaction can be stopped atany dimensionality as the nanomembrane extends from the primaryelectrode towards one or more secondary electrodes. The deposition alsocan be continued until the membrane contacts and forms an electricallyconducting bridge between the primary and the secondary electrode(s).The secondary electrode(s) used to guide the growth of the electroactivepolymer functionally in this instance becomes a primary electrode(s)once the nanomembrane becomes deposited on it.

An indirect, or coupled, electrochemical reaction also can be used tomanufacture nanomembranes on a primary electrode. In embodiments,indirect electrochemistries can include a 1,4-benzoquinone reaction (seeFormula I) provided by way of an example of the range of coupledelectrochemical reactions that can be employed to generate conditionsuseful for synthetic membrane formation. In this instance,electrochemistry can be used to react with 1,4-benzoquinone to produceionized hydrogen protons (i.e., creating a solution containing H⁺,thereby creating an acidic solution). The resulting protons in thesolution also can drive subsequent chemical reactions for nanomembraneformation in the instances in which monomers, ions, and/or doping agentshave been chosen which can be caused to form nanomembranes due to theformation of a localized acidic solution. It is understood that otherindirect electrochemical reactions similarly could be used to cause theformation of nanomembranes, that these electrochemistries can be drivenby primary and/or secondary electrodes, that they can involve the use ofcoatings and/or derivatization at either or both electrodes to keep thenanomembrane formation localized, and so forth.

Nanomembranes can have specific mechanical or durability properties. Inembodiments, they may be manufactured to be mechanically resistant orprone to a dimensionality change when being moved for example fromaqueous to dry environments, being moved between aqueous solutions ofdifferent ionic strength or pH, being moved between aqueous to organicsolvents, being subjected to shipping and/or prolonged storage, etc.

Electrochemically deposited nanomembranes can include one or morepolymers, metal materials, and/or other suitable materials that can beformed from the precursor materials by electrochemistries on electrodes.These different polymers and/or materials can be made in distinct layersand/or they can be comingled such that their distinctive properties aremore blended from one region of a nanomembrane to another. In oneembodiment, nanomembranes can be a polymer formed by polymerization froma precursor material of monomers due to direct/indirect electrochemicalreactions on suitable electrodes. In other embodiments, thenanomembrane(s) can be nanomembranes having metal materialelectrochemically accumulated on one or more primary electrodes fromprecursor materials, for example, but not limited to, metal ions and/ordoping agents.

To form a nanomembrane by electrochemical deposition, a reactionsolution, in embodiments, containing one or more precursor materialssuch as monomers and/or ionic materials, and/or doping agents, can beintroduced as a solution in contact with one or more primary electrodes.Solutions largely comprised of organic and/or aqueous solvents can beused to deliver the monomers, ionic materials, and/or doping agents. Byimposing electrical stimuli at the one or more primary electrodes, oneor more nanomembranes can be tunable manufactured by an electrochemicalaction thereon. Electrical stimuli imposed at one or more secondaryelectrodes also may be used to contribute to the deposition ofnanomembrane(s) on a primary electrode(s).

As disclosed herein, nanomembrane(s) can be tunably formed, e.g., in anelectrochemical deposition, by tuning various manufacturing parametersincluding, without limitation, the electrical stimuli used forelectrochemical reactions occurred on the electrode(s), number of cyclesof the electrochemical reactions, and reaction time for each cycle,selection of electrodes, selection of precursor materials, and otherpossible parameters during manufacturing. As a result, the tunablyformed nanomembrane(s) can have tunable amount, tunable dimensions,and/or tunable properties and functions (e.g., depending on selectedmonomers and/or metal ions) for the electrochemically depositedmaterial(s) on corresponding electrode(s).

Various precursor materials, in embodiments, monomers and/or derivatizedmonomers, and/or ionic materials and/or doping agents, can be chosen forthe properties that they impart by their chemical reactivity in theelectrochemistry reactions and/or for the properties and functions theyimpart on the resulting nanomembranes that they form. Non-limitingexamples of electroactive membrane precursor materials can include thefollowing without limitation:

etc.

Depending on the selection(s) of the Rn substituent(s) used, for exampleas provided in the in each of the above listed embodied materialsprovided herein, the resulting electrochemically deposited nanomembranescan be for example caused to have properties such as their beinghydrophobic, hydrophilic, charged, chemically reactive, metal-binding,metallic, and/or combinations thereof. In one embodiment, Rn can behydrogen. A wide spectrum of electrochemically driven reactions forforming polymers that can be nanomembranes as described herein are knownin the art. The following embodiments are provided as examples only andin no way are limiting to what can be accomplished byelectrochemistry-driven deposition reactions to form the apparatusherein.

Non-limiting examples of electroactive membrane precursor materials thatcan be acted on by direct and/or indirect electrochemical reactions togenerate a hydrophobic nanomembrane can include, in embodiments, polymerprecursor(s) with one or more substituents such as saturated and/orunsaturated hydrocarbons of sufficient carbon-chain lengths to provide agiven desired hydrophobicity. Such substituents can be the same ordifferent, and that they need not all confer hydrophobic properties, orthe same hydrophobic properties.

Non-limiting examples of precursor materials that can be acted on bydirect and/or indirect electrochemical reactions to generate ahydrophilic nanomembrane can include in embodiments, polymerprecursor(s) with one or more substituents such as hydroxyls, carboxylicacids, sulfonic acids, thiols, polyethylene glycols, and/or amines whichcan be used to provide a given desired hydrophilicity. Such substituentscan be the same or different, and that they need not all conferhydrophilic properties, or the same hydrophobic properties.

Non-limiting examples of precursor materials that can be acted on bydirect and/or indirect electrochemical reactions to generate a chargednanomembrane can include, in embodiments, polymer precursor(s) with oneor more substituents such as hydroxyls, carboxylic acids (and/or theirsalts), sulfonic acids (and/or their salts), thiols, and/or amines whichcan be used to provide a given desired charge due to their ionization,or lack thereof, when placed in a buffer of a buffer at a given pH. Inembodiments, amine-containing substituents in some types ofnanomembranes can be made to have a net positive charge in a givenbuffer, while carboxylic acid-containing sub stituents in some types ofnanomembranes can be made to have a net negative charge in the same orother buffers. Such substituents can be the same or different, and thatthey need not all confer the charge properties, or the same chargeproperties.

Non-limiting examples of electroactive nanomembrane precursor materialsthat can be acted on by direct and/or indirect electrochemical reactionsto generate a chemically reactive nanomembrane can, in embodiments,polymer precursor(s) with one or more substituents that can be reactivefunctional groups such as hydroxyls, carboxylic acids, esters andactivated esters, aldehydes , ketones, halogens, sulfonate esters,thiols, azides, alkenes, alkynes, phosphenes, and/or amines which can beused to provide a given desired chemical reactivity. Such substituentscan be the same or different, and that they need not all confer chemicalreactivity, or the same chemical reactivity. The resulting chemicallyreactive nanomembranes can further be used as an interface, inembodiments, functioning for attaching secondary molecules thereto.

Non-limiting examples of electroactive nanomembrane precursor materialsthat can be acted on by direct and/or indirect electrochemical reactionsto generate a metal ion binding nanomembrane can include, inembodiments, polymer precursor(s) with one or more substituents such asthiols which can be used to provide a given desired gold-ion (Au⁺)binding capability. Such substituents can be the same or different, andthat they need not all confer metal ion binding activity, or the samemetal ion binding activity.

Other non-limiting examples of precursor materials that can be acted onby direct and/or indirect electrochemical reactions to make metalizednanomembranes can include metal ions. The metal ions can be contained ina solution and then deposited onto an appropriately charged primaryelectrode such that a thickening metal layer forms on that electrode.Such a process is sometimes referred to in the art as being a form ofelectroplating. In embodiments, by applying appropriate electricalstimuli at the primary electrode(s), the electrochemically depositedmetal can form the nanomembranes. The disclosed metal deposition can bedriven with substantially the same apparatus and methods that arerequired for the electrochemical polymerization and/or nanomembraneelectroactivity reactions described herein. Possible difference withoutlimitation can be whether certain organic chemicals are included in agiven reagent formulation for synthetic membrane polymerizationreactions, or instead a given metal ion reagent formulation is used forelectrochemical deposition. In embodiments, metallic nanomembrane can bedeposited including, but not limited to, metal depositions such assilver, gold, platinum, titanium, copper, iron, tungsten, aluminum, etc.

In embodiments, doping agents also can be included in theelectrochemical or non-electrochemical deposition of nanomembranes. Thedoping agents can be added to a reaction mixture containing precursormaterials. The doping agents may or may not directly participate in theelectrochemical reaction and may just be ‘entrapped’ in any mannerwithin the resulting nanomembrane as it is being deposited. The dopingagents can include, but be not limited to, molecules that enhance orlimit the electrical conductivity of the nanomembrane (in embodiments,ions such H⁺, K⁺, SO₄ ⁻, Cl⁻, NO₃ ⁻, Cl⁻, I⁻, ClO₄ ⁻, PF6⁻, sulfonicacids, protic acids, benzoic acids, malonic acid, metal ions, metal ionsin chemical coordination elements such as cobaltabisdicarbollide);molecules that impart sites for chemical reactivity that are nototherwise present in the bulk nanomembrane (in embodiments, thiols,carboxylic acids, hydroxyls); molecules that cross-link or otherwiseincrease the mechanical strength or durability of the nanomembraneand/or increase or decrease the permeability of the nanomembrane toselective ions, hydrophobic/hydrophilic molecules, proteins, etc. (inembodiments, hydroxyethers, phenolic resins); and/or molecules whichincrease or decrease the ability of the nanomembrane to be effectivelyexamined via microscopy (in embodiments, for example, but not limited tothe use of doping with contrast agents such as metals and metal ions toaid in accommodating scanning and transmission electron microscopyexamination methods; doping with UV, visible light, infrared,fluorescent dyes, and/or quantum dots to aid in accommodating lightmicroscopy examination methods).

Note that electroactive nanomembranes deposited by means other thanelectrochemically-based methods also can include materials like thosedescribed that impart properties such as hydrophobicity, hydrophilicity,charge, chemical reactivity, and/or combinations of these. Inembodiments, these methods include, but are not limited to, methods suchas spraying, vapor-phase deposition, sputtering, spin coating,precipitation, in situ polymerization by electrochemical ornon-electrochemical means such as heat- and/or photo-curing, multilayerdeposition with the well layer being a sacrificial material that isetched away to create a material membrane over a well, etc.

Any combination of the above mentioned precursor materials, ionicmaterials, and/or doping agents, can be utilized to achieve the givendesired mechanical, chemical and/or electrical properties of resultingnanomembranes, whether deposited by electrochemical ornon-electrochemical means. Furthermore, these combinations may beemployed such that the properties are generally uniform within and/or atthe surface of a given nanomembrane. Nanomembranes also may be formedsuch that the properties are non-uniformly distributed within and/or attheir surface. As another non-limiting embodiment, a nanomembrane can bedeposited such that the portion of its deposited material that isclosest to the primary electrode(s) upon which it is formed can have onegeneral property in embodiments (e.g., being hydrophobicity, uncharged,and/or chemically unreactive), while the portion that is furthest awayfrom the primary electrode(s) has a different property (e.g., beinghydrophilic, charged and/or chemically reactive). In embodiments,non-uniform distributions of nanomembrane properties can be formed in,but are not limited to, layers, patches, gradients, etc. Furthermore,these properties can be made to change in their distribution and/orqualities over time via, for example but not limited to: imposingelectrical stimuli that results in the electrochemically-drivenaddition, removal, and/or modification of the nanomembrane's material;adding or removing material from the nanomembrane via coatings,derivatizations, and/or extractions of materials from the depositedpolymer; imposing electrical stimuli via one or more primary and/orsecondary electrodes so that the nanomembrane(s) structure changes indimensionality and a patch or layer of material possessing a givenquality is covered up or becomes exposed.

The nanomembranes, whether deposited by electrochemical ornon-electrochemical means, can be deposited to have electricalproperties, for example, providing electrical conductivity; capable ofdriving an electrochemical reaction that enables molecule attachment viachemistries; capable of driving indirect chemical reactions, etc.Specifically, electrically conductive nanomembranes can allow passage ofelectrons in a manner that is functionally similar to the way thatelectrons flow through a metal wire. This property may allow thenanomembranes to be used as an electric sensor surface for sensing thenanomembrane or for sensing at subsection(s) of the nanomembrane such asat points or projections on the surface, for sensing at growth-plates ofa nanomembrane's surface, etc. Nanomembranes can be capable of drivingan electrochemical reaction that enables molecule attachment viachemistries including oxidation and/or reduction reactions of materialsfor functionalization.

Nanomembranes formed to be capable of driving indirect chemicalreactions of, e.g., 1,4-benzoquinone reaction, can be provided by way ofthat enable production of acidic or basic solutions at specific placesalong a nanomembrane's surface. These acidic or basic solutions can inturn be used to catalyze subsequent chemical reactions, including butnot limited to, the attachment of molecules such as proteins, enzymes,antibodies, lectins, peptides, amino acids, modified amino acids,lipids, nucleic acids (single and double stranded DNAs, RNAs, aptamers),nucleic acid components (nucleosides, nucleotides, methylated/modifiedversions of the same), ionized salts, small molecules, drugs, etc, andcombinations thereof. These acidic and basic solutions also in turn canbe used to catalyze subsequent chemical reactions, including but notlimited to, the attachment of crosslinking agents on these points of ananomembrane's surface, which in turn can be used as points ofattachment for other molecules.

Nanomembranes can be synthesized to be relatively hydrophobic such thatmaterials like lipids can associate with them, and/or they can supportformation of a lipid layer (a monolayer or a bilayer) across ananomembrane's nanochannel. Hydrophobic nanomembranes can enableinsertion of, e.g., proteins, to form membrane-associated proteins.

Nanomembranes can be synthesized that chelate, coordinate, and/or bindmetal ions such that the nanomembrane becomes metalized. Following this,an electrical current can be applied via one or more primary electrodesupon which it is formed, and/or via one or more secondary electrodes, sothat electrical stimuli can be imposed on the nanomembrane. Theseelectrical stimuli can be used to drive other chemical reactions.Furthermore, the nanomembrane material can be chosen such that itsstructure is not disrupted by imposed electrical stimuli. Inembodiments, materials can be used for nanomembrane formation thatrequire high threshold electrical stimuli to induce a dimensional changeso that ‘lower’ electrical stimuli can be imposed on it without causingany dimensional change to it.

The disclosed device can include one or more nanomembranes formed ondesired electrodes of the nanowells/nanotubes. Each of the one or morenanomembranes can have the same or different desired properties andfunctions. The one or more nanomembranes can be generated bysimultaneous and/or sequential electrochemical or non-electrochemicaldepositions. The one or more nanomembranes can be either homogeneouslyor heterogeneously mixed in one single layer and/or non-uniform layers,regions, gradients, etc of nanomembranes during its formation. Forexample, a two-layer nanomembrane configuration can include a firstnanomembrane that is electrochemically or non-electrochemically formedon an electrode and a second electroactive nanomembrane formed on thefirst nanomembrane. Note that the number of materials and layers ofnanomembranes are not limited and any number of materials/layers and canbe formed on an electrode to provide desired properties, functions, andtunable nanochannels or nanopores formed by the electroactivenanomembrane.

In one embodiment, a method of forming multiple nanomembranes caninclude steps, for example, forming the first nanomembrane over theexemplary electrode using a first precursor material selected in anysuitable deposition processes, introducing a different precursormaterial, or replacing the first precursor material (that forms thefirst nanomembrane) with a second precursor material during a depositionprocess of the second electroactive nanomembrane. The secondelectroactive nanomembrane can have desired properties and functionsthat are the same or different than the first nanomembrane. Each of thefirst nanomembrane, the second electroactive nanomembrane, and/or anyadditional nanomembranes formed over the corresponding electrode can bethe same or different, for example, each can be hydrophobic,hydrophilic, chemically reactive, metal-binding, charge-binding(negative or positive ions), metallic, etc.

In one example, an exemplary device can include a first nanomembranethat is dense, non-conductive, and/or chemically unreactive. The secondelectroactive nanomembrane formed over the first nanomembrane and canbe, for example, porous, conductive, and/or chemically reactive, ascompared with the first nanomembrane. In another example,functionalizations can be performed to the surface of the electroactivenanomembrane of the formed nanochannel/nanopore.

In embodiments, nanomembranes are deposited such that the material thatis in close proximity to where a nanopore is positioned with it haschemical properties that are substantially different from the bulk ofthe rest of the nanomembrane. This can be accomplished by using thenon-electrochemical and/or non-electrochemical means. Non-limitingexamples of methods to accomplish this include employingnon-electrochemical methods such as spraying or sputtering of the ‘bulk’of a nanomembrane with one material and then adding a thin layer ofanother material to form a lip or edge that is in close proximity to thenanopore's opening. Electrochemical means also can be used to polymerizethe ‘bulk’ of a nanomembrane with one material, and then switching to adifferent material to electrochemically deposit a thin nanomembranecoating that creates the sidewall of a nanopore. The electroactiveproperties of the nanomembranes described herein also can be used, forexample imposing electrical stimuli on a nanomembrane(s) such that theirdimensionalities shift to form a nanopore with its sidewall composed ofmaterials that is/are functional different than the remaining ‘bulk’ ofthe nanomembrane. Combinations of electrochemical, non-electrochemical,and/or nanomembrane electroactivity induction methods are also possibleexamples of creating nanopore sidewalls that have functional propertiesthat are distinct from that of the bulk membrane. Regardless of themethod(s) used to create them, these types of nanomembrane nanopores canbe utilized in a range of embodiments. As one non-limiting embodiment,for example, this method can be used to provide for nanopores withsidewalls that are dimensionally tunable by imposed electrical stimuliwhile the bulk membrane is not dimensionally tunable. This approach canprovide particularly fine control over the electrical stimuli-induceddimensionality of a given nanopore's sidewall. In embodiments, thisapproach also can be used to localize functionalities such as a specifictype of chemical reactivity in close approximation to a given nanopore.A non-limiting example of this is to create a nanomembrane such thatchemical attachment points for functionalizations (e.g., antibodies,proteins, enzymes, nucleic acids, etc.) are located only in closeproximity to a given nanopore.

As described herein, electronic circuitry can be used to inducedimensional changes at a given deposited nanomembrane. Electroniccircuitry also can be utilized to electrochemically depositelectroactive material at a given primary electrode. In embodiments, thetunable manufacturing of nanomembranes with desired properties and/orfunctions also can be monitored during its fabrication by usingcircuitry that is electronically connected to one or more primary and/orsecondary electrodes. The electrode circuitry can be configured formonitoring the generation of the nanomembrane and/or monitoring the flowof analyte molecules (in embodiments, ionic material) through theresultant nanochannel in the nanomembrane. The monitoring process can becarried out by taking measurements via one or more primary and/orsecondary electrodes, including sensing through the nanomembrane itself.Such measurements can include, but are not limited to, measurements suchas current, resistance, voltage, capacitance, and/or combinationsthereof. Measurements also may be made to monitor the accumulation ofionic material and/or any other constellation of molecules at theelectrode(s) and/or at the nanomembrane itself, including monitoring formolecules that bind to and/or react with the nanomembrane(s) and/or oneor more primary and/or secondary electrodes in the apparatus. Inembodiments, by applying a voltage or a current to one or more primaryand/or secondary electrodes, the electrical resistivity provided by agiven nanomembrane can be measured. These measurements also can be usedto monitor and/or control the electrochemical deposition process anddimensionalities of a given nanomembrane anytime during and/or aftertheir manufacture.

FIG. 1 is a schematic of a portion of an exemplary device including ananowell array according to various embodiments of the presentteachings. The nanowell array in FIG. 1 can include a plurality ofnanowells 105 or nanotubes when the bottom of the nanowell is notincluded. The nanowells 105 can be provided in a solid support material108, for example, a semiconductor solid support material or any suitablesemiconductor materials used for an integrated circuit (IC) device,where standard semiconductor manufacturing techniques can be generallyused. The support material 108 can be suitable for integratingnanowells, nanotubes, nanomembranes, and/or bio-species therewith. Thesemiconductor material 108 can have a rigid or semi-rigid surface orsurfaces 109. One or more sidewall electrode(s) 130, 132, 134, 136 areincluded for an exemplary nanowell 105, which can further include abottom electrode 112, for example.

FIGS. 2A-2C are schematics showing nanomembrane deposition on sidewallof an exemplary nanowell according to various embodiments of the presentteachings. As shown in FIG. 2A, the device 200A can include an exemplarynanowell having one sidewall electrode deposited with a nanomembrane240. The nanomembrane can be deposited with various material depositiontechniques as disclosed herein. The deposition can beelectrochemically-induced or non-electrochemically-induced. Thenanomembrane 240 can be an electroactive nanomembrane regardless of thedeposition techniques used. In FIG. 2B, the nanomembrane 240 can furtherbe deposited having desired thickness on the associated electrode. InFIG. 2C, a second nanomembrane layer 260 can be, for example,electrochemically deposited over the first nanomembrane 240 in responseto suitable electrical stimuli such as a voltage or current applied tothe associated electrode or otherwise applied to the first nanomembrane240. In embodiments, this second nanomembrane layer can, for example, becomposed of a material that has distinct properties compared to thefirst layer of nanomembrane. It also, for example, can be a layer thatforms a thin coating as the sidewall of a given nanopore.

The disclosed apparatus/devices and methods can be used for a wide rangeof applications. The following only are examples of such applications,and these in no way limit the range of other applications for which theapparatus and/or methods provided herein.

As used herein, an “analyte” is one or more molecules included, but notlimited to molecules such as proteins, enzymes, antibodies, lectins,peptides, amino acids, modified amino acids, lipids, nucleic acids(single and double stranded DNAs, RNAs, aptamers), nucleic acidcomponents (nucleosides, nucleotides, methylated/modified versions ofthe same), ionized salts, small molecules, drugs, and/or combinationsthereof. Larger analytes are also possible including quantum dots,beads, particles, vesicles, liposomes, subcellular particles (e.g.,nuclei, endosomes, mitochondria, endoplasmic reticulum, lysosomes,Golgi, ribosomes, protein complexes, protein/nucleic acid complexes)cells, viruses, bacteria, mold, fungi, and/or combinations thereof,without limitation to the resulting dimensionalities of the assemblages.Analytes can be provided in a wide spectrum of formulations. Inembodiments, they can be provided in organic and/or aqueous solutions,in gases, in/on particles, etc., in mixtures, and/or combinationsthereof.

In one set of embodiments, the apparatus and methods described hereincan be used without limitation for applications that include ones whichenable the preparation, separation, partitioning, filtration, exclusion,inclusion, combination, reaction, binding, unbinding, isolation, etc. ofone or more analytes, processes which can be carried out separatelyand/or in combination and which are collectively referred to herein withrespect to analytes-related processes as being analyte “interactions”with nanomembranes. Thus, analytes generally are described herein withthe terminology that they are being caused to interact with ananomembrane. However, it should be understood that analytes can in factbe caused to interact with any number of nanomembranes at any given timeaccording to the same terminology. Similarly, it should be understoodthat analytes can be caused to “move” within a nanochannel as definedherein, whether this nanochannel contain a nanomembrane(s) or not, andthat this same terminology also describes that analytes can be caused to“move” in within any number of nanochannels over a given time.

Analytes and other molecules can be made to “move” a nanochannel by anumber of methods. In embodiments, the molecular motion of analytes canbe passive Brownian motion-driven diffusion. Such passive diffusiongenerally can be enhanced or slowed by raising or lowering thetemperature of the solution or gas containing the analyte(s),respectively. Temperature control can be provided by heating and/orcooling elements on or around a solid support(s), chamber, pumpingsystem, environmental control system, etc. employed for givenapplication.

It also is possible in embodiments to impose electrical stimuli such as,but without limitation, a voltage or current across a given nanomembranesuch that analytes and other molecules are subjected to electrophoreticforces that can move them towards, away from, and/or through a givennanochannel. For analytes and other molecules to be electrophoresedunder such conditions, they need to bear at least a transient or netcharge, either positive or negative in sign such that they are attractedto the electrode(s) with an opposite polarity and repelled from theelectrode(s) of the same polarity. Such analyte motion can for examplebe created by electrically polarizing one or more of the availableelectrodes “above” a given nanomembrane in a nanochannel such that it,or they, are opposite in polarity (i.e., to be positive or negative insign, such as to serve as a cathode or anode) to the polarity of the oneor more electrodes below this nanomembrane. The same or differentvoltage can be imparted on each nanochannel's nanomembrane(s) asdesired, even as they occur in a given nanotube and/or nanomembrane.Such electrical stimuli may be invariant and/or may vary in wavedimensionality, amplitude, frequency, duration, etc., and combinationsthereof. These variations may be recurrent, or may also vary over time.The analyte motion-inducing electrical stimuli may be utilized to causethe electrophoresis of the exemplary analyte(s) into or out of a givennanochannel at a given rate, for example, but without limitation, byapplying a higher bias voltage to cause a given type of analyte to moveinto or out of the nanochannel(s) at a faster rate than can beaccomplished by the application of a lower bias voltage. Analytemotion-driving electrical stimuli also can be transiently alternated insign to reverse the course of an ionized molecule, which has becomelodged in, or plugged a nanochannel. Similarly, such electrical stimulican be reversed for a period of time, for example to drive ionizedmolecules out of a nanowell to diminish a concentration gradient formedthere following a period in which such ions previously have beenelectrophoresed into a given nanowell. Likewise, such electrical stimulican be reversed for a period of time to drive ionized molecules from oneor more chambers connected by a given nanotube such that they are movedto the one or more other chambers at the other end of a given nanotube.Note, too, that a electrical stimuli-driven analyte movement also can beused to move analytes within a given nanochannel. In embodiments,imposed electrical stimuli can be utilized to move molecules from thebottom of a nanowell to one or more sidewall electrodes, to movemolecules from one portion of a sidewall to another, to move moleculesfrom one nanotube to another, from one nanotube to one or morenanowells, etc. The apparatus and methods in the art regarding capillaryelectrophoresis, capillary zone electrophoresis, and gel electrophoresiscan be utilized to enable these types of embodiments to move analytes byelectrophoresis. These electrophoretic forces also can be utilized to‘push’ or ‘pull’ analytes and other molecules from within one or morechambers and into one or more nanochannels. For example, in embodiments,electrical stimuli can be imposed at one or more bias electrodes suchthat these stimuli are ‘paired’ with one or more sidewall or bottomelectrodes in the case of a nanowell, or one or more sidewall or biaselectrodes at the opposite end of a nanotube in the case of a nanotube.Such a pairing including imposing electrical stimuli such that themembers of the pairing are opposite in sign (i.e., the electrode(s)including one ‘side’ of the pairing has a positive sign/polarity, whilethe other electrode(s) including the other side of the pairing has anegative sign/polarity). Such a pairing can be utilized to attract orrepel ionized analytes possessing a positive or negative charge togenerally flow towards electrode(s) side of the pairing of the oppositesign/polarity. This embodiment describing the ability to attract orrepel charged analytes and other molecules towards or away fromnanochannels, whether containing nanomembranes or not, is referred toherein as using imposed electrical stimuli to create an “ion funnelcurrent/voltage.” In the embodiments in which an ion funnelcurrent/voltage is also being utilized to draw analytes and othermolecules from a chamber and past or through a given nanomembrane, oneof the electrode(s) comprising an ion funnel current/voltage pairing isplaced ‘above’ the nanomembrane and the other electrode(s) comprisingthe other ion funnel current/voltage pair is placed ‘below’ thenanomembrane.

A wide spectrum of technologies in the art also can be used to generatemechanical pressure to move analytes across the surface of, or through,a given nanomembrane. In embodiments, mechanical pressure that moveanalytes can be provided by the use of pumps, vacuums, pressurizedliquids, pressurized gases, centrifugation, etc., and combinationsthereof. The apparatus and methods in the art regarding liquidchromatography including low pressure, high performance/HPLC,filtration, and the like are generally appropriate to move analytes inliquids. In other embodiments, the apparatus and methods in the artregarding gas chromatography and the like are generally appropriate tomove analytes in gases.

Making use of the electrically tunable dimensionality capabilities of agiven nanomembrane is another means by which analytes can be movedacross its surface(s). In embodiments, a given nanomembrane can besubjected to oscillations of imposed electrical stimuli such that it‘stirs,’ or otherwise moves around in, a given solution or gas.Similarly, groups of nanomembranes can be subjected to imposedelectrical stimuli oscillations such that they generally ‘sweep’ orotherwise move analytes, including in a pulsatile, peristaltic, and/orwave-like fashion, across or through a given nanomembrane ornanochannel.

It should be understood that these modes by which analytes can beinduced to move through a nanochannel can be subjected to significantmodulation as desired. Thus, in embodiments, analyte movements withrespect to a given nanomembrane can be induced to be essentiallyunidirectional, bidirectional/reversible, random, etc., and combinationsthereof, and these movements also can be varied in their force,frequency and duration as is desired for a given application.Furthermore, in embodiments, analytes can be moved utilizing just one,or combinations of two or more, of the analyte motion-inducing apparatusand methods described herein.

In embodiments, a wide variety of combinations of one or moreelectrically tunable nanomembranes with one or more possible modes toinduce analyte motion into, within, and/or through a given nanochannelprovide for useful applications. These include, but are not limited by,the following examples.

In embodiments, one or more nanomembranes within a nanochannel can bearranged to form a barrier such that only analytes of a sufficientlysmall size can pass through the nanochannel that they form. Barrierscomprised of nanomembranes of a given charge (in embodiments, bearingpositively or negatively charged molecules) can also be constructed suchthat they only provide for a relatively unhindered passage of similarlycharged analytes, while analytes of a charge opposite to suchnanomembranes are retarded and/or rendered immobile. Similarly,nanomembranes can be constructed of a given degree of hydrophobicity orhydrophilicity such that they do or do not retard the flow of, orotherwise render immobile, molecules of an appropriate hydrophobicity orhydrophilicity.

Nanomembranes can be constructed in a given nanochannel and thensubjected to an appropriate imposed electrical stimuli at the primaryand/or secondary electrodes in and around the nanochannel such that apH/charge gradient is formed from one portion of the nanochannel toanother. Analytes moving within such a gradient, especially when underelectrophoretic forces, can become immobilized in the portion of thenanochannel in which their charge is effectively neutralized (i.e., whena given analyte moves to a position in the nanochannel that matches itsisoelectric point). The apparatus and methods in the art regardingisoelectric focusing can be utilized to enable these types ofembodiments.

A given nanomembrane also can be functionalized to bind passinganalytes. In embodiments, they can be functionalized with molecules suchas antibodies, antibody fragments, aptamers, lectins, phage, etc. suchthat passing analytes can be retarded, or rendered immobile, underappropriate conditions.

A given nanomembrane also can be modified with chemically reactivemolecules such that they act on an appropriate analyte as it passes. Inembodiments, a nanomembrane can be modified with enzymes, reactivechemicals, etc. such that they exert a chemical reaction upon passinganalytes under appropriate conditions.

A given nanomembrane can be constructed such that it carries out morethan one desired functionality, or combinations of functionalities, at agiven time. In embodiments, a nanomembrane can be constructed such thatit binds analytes of a given charge while it also blocks the passage ofanalytes over a given size. A great many other combinations offunctionalities are also possible in embodiments.

The apparatus and methods described herein also can be employed tomodify a given nanomembrane's tunable properties. In embodiments, agiven nanomembrane can be controllably altered in its dimensionality inreal-time, stepwise, and/or spread over a continuum or gradient suchthat molecules of increasingly larger or smaller size are allowed topass through a given nanochannel. Similarly, a given nanomembrane'scharging, hydrophobicity, hydrophilicity, etc., can be changed to affecthow analytes interact with it over time.

By changing the properties of a given nanomembrane in electricallytunable manner, it also is possible to unbind (elute) analytes that havebecome bound to it. Such methods can be gradated via electricalstimuli-induced changes that are stepwise, spread over a continuum orgradient, etc. such that they enable analytes to become unbound inspecific groupings and/or isolated from other groupings. In embodiments,such methods can include the induction of an electrochemical reactionsuch that analytes are unbound as a nanomembrane is caused todepolymerize. A nanomembrane also can be electrically induced to changeshape such that analytes are released.

The apparatus and methods described herein also can be used to causeanalytes to unbind via electrophoretic forces. In embodiments, byimposing electrical stimuli at the primary electrode(s) upon which thenanomembrane is deposited (i.e., electrophoretic forces that areprovided via conductance through a given nanomembrane) and/or one ormore secondary electrodes in the apparatus, analytes can be subjected tosufficient forces such that they become unbound from the nanomembrane.

Analytes also can be made to unbind from a given nanomembrane by theapplication of a suitable fluid to release them (or gas, or combinationsthereof). Charged analytes generally can be unbound from chargednanomembranes and/ or nanomembranes modified by proteins, aptamers, andthe like via the application of a solution containing ionized molecules(in embodiments, an aqueous solution containing dissolved salt).Similarly, analytes generally can be unbound from hydrophobic and/orhydrophilic nanomembranes via the application of a solution containinghydrophobic/hydrophilic molecule stabilizing/solubility agents (inembodiments, ethanol, dimethylsulfoxide, detergents, salts). Suchmethods can be gradated in embodiments, using agents that are addedstepwise, spread over a continuum or gradient, etc. such that theyenable analytes to become unbound in specific groupings and/or isolatedfrom other groupings. In embodiments, the apparatus and methods in theart regarding ion exchange chromatography, including low pressure, highperformance/HPLC, filtration, etc, in particular can be utilized toenable these types of applications in which charged analytes are beingunbound. In embodiments, the apparatus and methods in the art regardingreverse phase chromatography, including low pressure, highperformance/HPLC, filtration, etc., in particular can be utilized toenable these types of applications in which hydrophobic and/orhydrophilic analytes are being unbound.

Changing the temperature of a nanomembrane (or the solution or gas orcombinations thereof that are passing over the nanomembrane) can also beused to unbind some analytes. Such methods can be gradated inembodiments, via direct heating and/or electrical stimuli-inducedchanges that are stepwise, spread over a continuum or gradient, etc.such that they enable analytes to become unbound in specific groupingsand/or isolated from other groupings.

Changing the mechanical pressure applied to a given nanomembrane alsocan be utilized to unbind analytes from a given nanomembrane. This canbe accomplished by adjusting the settings on the pumps, vacuums, etc.that are used to generate mechanical pressure in order to increase ordecrease the pressure being applied on a given nanomembrane.

A wide variety of apparatus and methods can be employed to monitor theanalytes that are applied to, pass through, are bound by, and/or areunbound from a given nanomembrane described herein.

Analyte interactions with a given nanomembrane, including all of thetransients and/or binding interactions described herein, can bemonitored directly by the apparatus and methods described herein inembodiments. In one type of embodiment, the electrode circuitry for oneor more primary and/or secondary electrodes can be configured to monitorchanges in the electrochemical capabilities and/or tunabledimensionalities of a given nanomembrane that result from interactionswith analytes (e.g., in embodiments to monitor changes in an imposedelectrical stimuli's ability to induce a modification in a givennanomembrane's polymerization, depolymerization, composition change,dimensionality, etc. as described herein, that indicates ananomembrane's interactions with analytes will, are, or have beenoccurring). The interaction of analytes also can be monitoring viataking measurements through one or more electrode(s) in the solidsupport, including sensing through the nanomembrane itself, and/or viaone or more electrodes located elsewhere (i.e., in a chamber). Inembodiments, analyte interaction monitoring modes at a givennanomembrane can include, but are not limited to, measurements such ascurrent, resistance, voltage, and/or capacitance through thenanomembrane and/or at one or more of any sidewall, bottom, bias,primary, and/or secondary electrodes in the apparatus, and/or anycombinations of the above. The monitoring of analytes via theaccumulation of charged analytes, ions and/or any other constellation ofmolecules at one or more electrodes in the apparatus and/or at a givennanomembrane itself also are possible in embodiments. In embodiments,monitoring also can be conducted via the reaction of analytes and/or anyother constellation of molecules at one or more electrode in theapparatus, and/or at a given nanomembrane. By imposing electricalstimuli to one or more electrodes in an apparatus, the electricalresistivity of a given nanomembrane interacting with analytes can bemeasured in related embodiments. The flow of analytes as determined bymodulations in the requirements of an apparatus' electronics to providefor an “ion funnel current/voltage” through a nanomembrane's nanopore isanother means by which analytes can be monitored for their nanomembraneinteractions in embodiments.

In embodiments, one or more electrodes made of, or covered by, silver isplaced ‘below’ a nanomembrane(s) past which an ion funnelcurrent/voltage is imposed. The ion funnel current/voltage is thenconfigured to drive negatively charged chloride (Cl—) ions ‘down’towards the silver electrode(s). As the chloride ions contact the silversurface, the well-known silver/silver chloride reaction can ensue suchthat an electron density develops at that electrode. The development ofthis electron density can be monitored for example, but not limited by,the numerous current and/or voltage measuring devices that also are wellknown in the art. The ion funnel current/voltage also can drivenegatively charged analytes ‘down’ past the nanomembranes, andpositively charged analytes ‘up’ past the nanomembranes. The flow ofnegatively and/or positively charged analytes can affect the movementrate, and/or count, of chloride ions that are driven towards the silverelectrode over a period of time, particularly as might be crowdedtogether in the confined space of a nanochannel or nanopore. Inembodiments, these effects can result in characteristic signalmodulations that have, for example, been used to identify specific typesof DNA nucleotides as they flowed sequentially through a proteinnanopore (Clark et al., Nat. Nanotechnol 2009). Thus, thischloride/silver/silver chloride measuring system enables one particularmode of embodiments for measuring the flow of analytes pastnanomembranes.

A wide spectrum of other instrumentation and methods, which can be usedin combination(s) with the apparatus and methods described herein, alsocan be used to monitor analytes interacting with a given nanomembrane.For example, samples of an analyte formulation can be taken before andafter they have been applied to the nanomembrane-bearing apparatusdescribed herein to determine what analyte(s) may have been removed,added, or remain unchanged in concentration and/or composition as aconsequence of their interactions within the nanomembrane. Such samplingin embodiments can be carried out by extracting materials from the oneor more chambers ‘before’ and ‘after’ an analyte formulation has beenmoved through one or more nanomembrane-deposited nanochannels innanotubes, and/or by extracting materials from ‘above’ and ‘below’ oneor more nanomembrane-deposited nanochannels. In embodiments, samplesalso can be taken from within a given nanochannel, including sampling agiven nanomembrane in the nanochannel. In embodiments, samples can betaken from the material comprising the electrode(s), solid support(s),chambers, and/or sidewalls in a given apparatus. In embodiments, samplesalso can be taken from any of the gas- and/or fluid-filled spaces in agiven apparatus (i.e., taking samples from the spaces in the chamber(s),nanowell(s), and/or the nanochannel(s) of an apparatus). The sampling ofthese spaces can be carried out via one or more ports manufactured toprovide access to them, and/or by gaining access by force through agiven chamber wall, solid support, sidewall, and/or nanowell bottom. Inany case, analytes can be analyzed for their composition and/orquantitated via whichever of the many well-described instruments andmethods in the art are most appropriate. For example, in embodiments,such analyte monitoring instruments can include, but are not limited to:spectroscopy (nuclear magnetic resonance, Raman, surfaced enhancedRaman, fluorescence, ultraviolet, luminescence, visible light, X-ray,X-ray photoelectron, infrared, terahertz, atomic force), flameionization, surface plasmon resonance, surface acoustic wave,antibody-based, ELISA, peptide-based, lectin-based, enzyme-based,peptide/protein sequencing, nucleic acid/oligomer sequencing,lipid-based, small molecule-based, optical wave guide-based, MEMS-based,pulsed amphometric detection, nephelometry, circular dichroism, X-raydiffraction, radiochemical detection, mass spectrometry, and/orcombinations thereof.

The electroactive nature of the nanomembrane provided herein alsoenables embodiments for the directly tunable, real-time control over thecross-sectional area of a nanochannel that is occupied by a givennanomembrane in a nanotube or nanowell. One embodiment for suchnanomembranes is that they can be employed as real-time dimensionallytunable structures to create size-oriented barriers and/or sieves foranalytes that can be made to move around, over, and/or through thenanomembrane(s) surface. As one non-limiting example of this embodiment,one or more nanomembranes can be deposited in a nanotube such that theyinitially allow only comparatively small analytes (e.g., small ions,nucleic acids, amino acids, solvated small molecule drugs, etc.) to moveunhindered through the nanochannel. By imposing appropriate electricalstimuli on the nanomembrane(s), a dimensional change can be induced inreal-time such that now larger analytes (e.g., peptides, nucleic acidoligomers, etc.) can be caused to move unhindered through thenanochannel. By imposing yet other appropriate electrical stimuli on thenanomembrane(s), now even larger analytes (proteins, aptamers, etc.) canbe moved through the nanochannel. Thus, the apparatus described hereincan be utilized to create a device that separates analytes according totheir size, and that this capability can be modulated in real-time.

Dimensional changes also can include changes in the molecularcomposition of a given nanomembrane can according to the apparatus andmethods described herein. For example, in embodiments, electrochemicalreactions can be enabled in real-time by applying electrical stimuli atone or more primary or secondary electrodes such that a givennanomembrane is subjected to a polymerization and/or depolymerizationreaction(s). A given nanomembrane under such electrical stimuli controlsthereby in embodiments can be induced to add or remove molecules, changein its charge, expose/hide portions of itself, etc. such that itssurface and/or internal composition can be change in real-time. Suchreactions under induced electrical stimuli control, in embodiments, cancause the resulting nanomembrane to become more hydrophobic,hydrophilic, charged, metal binding, and/or combinations thereof. Directand/or indirect electrochemical reactions also can be utilized to attachor detach materials from the nanomembrane, including functionalizationmaterials such as proteins, enzymes, antibodies, lectins, peptides,amino acids, modified amino acids, lipids, nucleic acids (single anddouble stranded DNAs, RNAs, aptamers), nucleic acid components(nucleosides, nucleotides, methylated/modified versions of the same),ionized salts; small molecules, drugs, and/or combinations thereof. Asone non-limiting embodiment of this, one or more nanomembranes can bedeposited in a nanotube such that they initially bind to molecules thathave low-to-high negative charge densities. This in a non-limitingexample can be accomplished by depositing nanomembrane material(s) thatinitially bears a dense positive charge at its surface and/or anyinternally analyte-accessible portions. Analytes of a low-to-highnegative charge (for example, but not limited to, small to large nucleicacid oligomers) can bind to this nanomembrane(s) as they are movedthrough the nanochannel, while positively charged analytes remainunbound and move relatively unhindered through the nanochannel. Byimposing appropriate electrical stimuli on the nanomembrane(s), adimensional change can be induced such the nanomembrane(s) looses asignificant portion of its positive charge density (for example, but notlimited to, depolymerizing aspect of the nanomembrane that bear positivecharge, undergoing an electrochemical reaction that eliminates positivecharges without requiring membrane depolymerization, and/or undergoingthree-dimensional change that covers up a portion of the nanomembrane'spositive charge). This change can result in the analytes withcomparatively less negative charge (for example, but not limited to,small(er) nucleic acid oligomers) becoming unbound by thenanomembrane(s) such that they now can be made to move unhinderedthrough the nanochannel. By imposing yet other appropriate electricalstimuli on the nanomembrane(s), now analytes bearing even highernegative charges (for example, but not limited to, large(er) nucleicacid oligomers) become unbound and can be moved through the nanochannel.In another embodiment, a nanomembrane can be deposited such that thesame overall methods can be used to separate analytes based on theirpositive charge using nanomembranes comprised of materials that can bemodulated in their negative charge density. Thus, the apparatusdescribed herein can be utilized to create a device that separates,isolates, extracts, etc. analytes according to their positive ornegative charge, and that this capability can be modulated in real-time.

In other embodiments, a similar approach can be to create a device thatenable analytes to be separated/isolated/purified/extracted/etc. byhydrophobicity, hydrophilicity, functionalization binding, metalbinding, chemical reactivity, and properties, too, and be modulated inreal time.

In still other embodiments, a similar approach can be undertaken toenable analytes to be separated/isolated/purified/extracted/etc. by twoor more of their properties such as their hydrophobicity,hydrophilicity, functionalization binding, metal binding, chemicalreactivity, and the like. As one non-limiting example of this,nanomembranes can be deposited so that molecules such as nucleic acidoligomers, antibodies, aptamers, lectins, etc. functionalize them,thereby enabling the binding of analytes of an appropriate type to thatnanomembrane. The same nanomembranes can be deposited in a manner suchthat they at least initially only allow comparatively small analytes tomove through the nanochannel(s) with which they are associated. When inuse, the nanomembranes in such a device can then be tuned in real timesuch that they increase or decrease their properties for binding ofgiven analytes even as they are tuned to permit or restrict the passageof analytes according to their size. Other non-limiting embodimentsinclude nanomembranes formed to interact with analytes in combinationssuch as chemical reactivity and size, charge and size, functionalizationand charge, functionalization and hydrophobicity/hydrophilicity,chemical reactivity and charge, and the like.

In another set of embodiments, one or more nanomembranes can beconstructed in a nanochannel such that it/they support the formation,stability, and/or destabilization of one or more lipid bilayers. Theseembodiments also can be used to exclude the incorporation of moleculesinto lipid bilayers based upon molecular size.

As described herein, “lipid bilayers” generally are composed of lipids(e.g., phospholipids, fatty acids, glycerides, etc.). When thesemolecules are appropriately combined, they can spontaneouslyself-assemble into a roughly two-layered structure. In this structure,the lipid molecules are aligned on both sides of the layers so that havetheir hydrophobic ‘tails’ oriented towards the bilayer's interior andthey have their hydrophilic ‘head groups’ oriented towards the outerfaces of the bilayer. Lipophilic molecules also can be included in, orassociated with the surface(s) of lipid bilayers (e.g., cholesterols,amphipathic proteins, hydrophobic molecules, etc. can be ‘dissolved’within a given bilayer; various charge-binding molecules, etc. can becoordinated by, or bound to, the lipid head group(s) at the surface of abilayer).

Lipid bilayers are used for a wide variety of apparatuses and methods inthe art, for example ones making use of their ability to provide for abarrier between two aqueous compartments for hydrophilic molecules, toprovide for an electrically resistive barrier between two compartments,etc. Lipid bilayers also utilized to provide an artificial structureinto which certain membrane proteins can be inserted and tested fortheir functionalities. Important classes of such membrane proteinsinclude the porins, ion channels/complexes, and protein nanopores. Notethat these protein nanopores are distinct from the “nanopores” asdefined herein. Protein nanopores are composed of one or more proteinswhile “nanopores” as defied herein are created by encirclingnanomembrane sidewalls. In order to test their functionality, thesemembrane proteins are generally first inserted into a lipid bilayer.They often then are treated by the addition of a molecule to one side ofthe lipid bilayer to determine whether this molecule augments orinhibits a basic feature of the membrane protein's functionality (e.g.,to determine whether the molecule is an agonist or an antagonist of themembrane protein). A membrane protein's functionality is determined, forexample, by how it effects the electrical and/or analyte permeabilitythrough a lipid bilayer and/or through the bilayer-spanning proteinnanopore formed by a given membrane protein. For example, lipidbylayer-embeded α-hemolysin protein nanopores have been used as tomeasure the length of single-stranded DNA molecules (Ayub et al., J.Phys.: Condens. Matter 22, (2010) 454128). Similar systems also havebeen used to monitor the flow of DNA oligomers through protein nanoporesembedded in lipid bilayers (Clark et al., Nat. Nanotechnol 2009).Nonetheless, the utility and robustness of these membrane proteinstudies are considerably challenged by the generally instability oflipid bilayers. In particular, lipid bilayers are easily disrupted bymechanical shock, thermal motion, and/or the presence of even traceamounts of detergents or organic solvents. Conventional approaches forovercoming these challenges include making use of solid-state nanoporesformed by semiconductor fabrication techniques involving focusedelectron and ion beams to create nanopores in a semiconductor material.

Embodiments of the apparatus and methods described herein can be used tocreate a greatly stabilized lipid bilayer. Lipid bilayer also can betuned in their dimensionalities by the electroactive nanomembranesdescribed herein such that they can be made to size-exclude moleculesfrom inserting into their surfaces.

A general feature of these embodiments is that they include one or morenanomembranes being deposited on a given nanochannel's sidewall, andwhich are generally hydrophobic in the characteristics of theirsurface(s). Such hydrophobic surfaces can serve as a site or sites thatenhance the formation of (e.g., they serve as bilayer nucleation sites),and/or lipid adhesion sites such that the resulting lipid bilayer ismore strongly tethered to a nanochannel's sidewall than that it would bewithout the presence of the nanomembrane(s). The use of encirclingnanomembranes (i.e., ones forming a nanopore as described herein) can beparticularly useful in supporting a given lipid bilayers formation andsubsequent stabilization. In embodiments, it can be useful to cause theformation of more than one lipid bilayer in a given nanochannel usingone or more nanomembranes.

Another general feature of these embodiments is that the electroactivetunable dimensionality of the sidewall nanomembrane(s) enables them tobe modified such that it decreases the total surface area that must bespanned by a given lipid bilayer. Lipid bilayers of smaller surfaceareas are known to be more stable than larger ones, so enablingnanomembranes to be tuned to provide for just enough lipid bilayersurface area to be formed to support a given application promotes theiroverall utility.

Utilizing the dimensional tunable selection of a nanomembrane-supportedlipid bilayer also has the utility of providing a size-selectivity formolecules that can be inserted in a given lipid bilayer. In suchembodiments, nanomembranes, and particularly encircling nanomembranethat create a nanopore as described herein, can be formed such that theysupport and stabilize the formation of a size-restricted lipid bilayerfor a surface area that allows for the insertion of only a small number,or even just one, membrane protein, into the bilayer. Non-limitingembodiments include lipid bilayer-stabilizing a nanomembrane nanoporesformed to stabilize a lipid bilayer such that it is size-restricted sothat just one membrane protein, for example but not limited to, proteinporins, ion channels, or protein nanopores, can insert into the lipidbilayer.

In some embodiments, it can be useful to disrupt a given lipid bilayerformed in a nanomembrane(s) nanochannel. In embodiments, electricalstimuli can be applied to such a nanomembrane to depolymerize,polymerize and/or otherwise change its dimensionality such that thelipid bilayer is effectively disrupted. Electrochemical depolarizationand chemical reactivity methods also can be used in embodiments to makea nanomembrane become too hydrophilic to support a bilayer byelectrochemically shielding and/or reacting away the nanomembrane'shydrophobic surface. One or more primary and/or secondary electrodes inembodiments also can be subjected to imposed electrical stimuli suchthat a lipid bilayer is disrupted by the attraction, repulsion, and/orelectrophoresis of charged molecules in a lipid bilayer. Theelectrophoretic methods described herein also can be utilized to push orpull apart a lipid bilayer by asserting electromotive force(s) on thecharged molecules of, and around, the lipid bilayer. The instruments andmethods to induce mechanical pressure and/or temperature changesdescribed herein also can be used in embodiments to disrupt a givenlipid bilayer. In addition, combinations of two or more of thesemethods, oscillating these methods over time, etc. can be utilized todisrupt a nanomembrane-stabilized lipid bilayer.

The functionality of a lipid bilayer and/or molecules contained withinit for such embodiments can be evaluated directly by the apparatus andmethods described herein. In one type of embodiment, the electrodecircuitry for one or more primary and/or secondary electrodes can beconfigured to monitor changes in the electrochemical permeability,resistivity, etc. of the lipid bilayer and/or analytes therein, and/orthe analytes passing through the lipid bilayer (as well as throughprotein nanopores therein). These electrode(s) measurements can includesensing through the nanomembrane itself via the primary electrode uponwhich the nanomembrane is deposited, and/or via one or more electrodeslocated elsewhere. In embodiments, the measurements made can include,but are not limited to, measurements such as current, resistance,voltage, and/or capacitance through the nanomembrane and/or at one ormore of any sidewall, bottom, bias, primary, and/or secondary electrodesin the apparatus, and/or any combinations of the above. The monitoringof an accumulation of charged analytes, ions and/or any otherconstellation of analytes passing through a lipid bilayer, and/orthrough a protein nanopore within a lipid bilayer, can be made via oneor more electrodes in that are possible in embodiments. In embodiments,monitoring also can be conducted via the reaction of analytes and/or anyother constellation of molecules passing through a given lipid bilayer,and/or through a protein nanopore within a lipid bilayer, via the use ofone or more electrode in the apparatus, and/or at a given nanomembrane.

One particular set of embodiments, but not limiting others, is tomonitor the functionality of membrane proteins, including, but notlimited to: porins, ion channels, protein nanopores, membrane proteins,membrane enzymes, membrane receptors, membrane channel proteins,membrane transport proteins, complexes with and by thereof, andcombinations thereof. Several of these membrane proteins, such as theporins as ion channels, are well known to form pores through which ionscan be restricted, gated, and/or allowed to pass from one side of alipid bilayer to the other by opening and closing in a regulated mannerin response to agonists and antagonists of their functionalities. Inembodiments, one or more of these proteins can be inserted into a givennanomembrane-supported lipid bilayer. In particular, a nanomembranewhich provides for a size-restricting lipid bilayer that generallyprevents more than one of these membrane proteins to be inserted in thelipid bilayer is preferred. The functionality of membrane proteins suchas the porins and ion channels can be tested by placing a given testmolecule (e.g., a known or potential agonist and/or antagonist of theprotein's functionality) into the solution ‘above’ a given proteininserted into a lipid-bearing nanomembrane. A change in the membraneprotein's functionality is then measured by one of the analytemonitoring methods described herein. In particular embodiments,monitoring is carried out using the apparatus and methods forelectrode-based ion flow analyte monitoring provided herein. Theapparatus and methods described herein to create an ion funnelcurrent/voltage that draws analytes to the protein in a lipid bilayer,and/or to further manipulate the flow of ions through the opened poresof these proteins also are embodiments. As one non-limiting example, thechloride/silver/silver chloride measuring system described herein can beutilized to monitor the flow of ionic materials in these embodiments,for example to monitor the opening and/or closing of a given proteinnanopore under the influence of the presence of agonists and/orantagonists. Other non-limiting embodiments, though, includefunctionality assays for any other of the lipophilic molecules, membraneprotein, and membrane protein complexes.

Another particular set of embodiments enables the application of nucleicacid sequencing. In these embodiments, a protein nanopore (for example,but not limited to α-hemolysin or Mycobacterium smegmatis porin Ananopores) is inserted into a given nanomembrane-stabilized lipidbilayer. A size-restricting lipid bilayer that generally prevents morethan one of these membrane proteins to be inserted is preferred. Nucleicacids are then placed in the solution in the chamber(s) ‘above’ thenanopore(s) in the lipid bilayer. The nucleic acids can be singlestranded DNA, or RNA, oligomers of any length. These nucleic acidoligomers may or may not contain naturally or artificially modifiednucleotides (e.g., nucleic acids naturally modified such as containing5-methylcytidine; nucleic acids artificially modified such as to insertspacers between the nucleotides and/or to modify the nucleic acid'sbases with ‘tags’ to increase their bulk, and/or to modify theirproperties when monitored by any given sensor system, etc.). The nucleicacid oligomers also may be double stranded, or partially doublestranded, oligomers of any length. In the instance of the oligomer beingpartially double stranded, such an oligomer can for example be formed byhybridizing a complementary nucleic acid oligomer of any length so longas it is sufficiently stable to maintain its double-stranded form in auseful manner for testing (e.g., complementary oligomers made of twonucleotides or longer and of know sequence are used in applications inthe art to create partially double stranded oligomers). After adding thenucleic acid oligomer into the solution ‘above’ the protein nanopore, anion funnel current/voltage as described herein can be initiated to causethe negatively charged nucleic acids to move towards, and eventuallythrough, a given protein nanopore. Deriving the sequence of the nucleicacid as it passes through a given protein nanopore as it is residing ina nanomembrane-stabilized lipid bilayer can be carried out by severalapplications of the methods and apparatus described herein. Whatevermode is used to identify the oligomer's sequence, though, theoverarching process of obtaining an oligomers sequence as it passesthrough a protein nanopore (or through a nanomembrane-encircled nanoporeor suitable nanochannel), this process generally is described in the artas ‘sequencing-by-threading’ since it requires a given oligomer to bepassed through a hole/pore that metaphorically is akin to passing athread to the eye of a needle. In one set of embodiments, the flow ofions passing through the protein nanopore along with a nucleic acidoligomers is monitored using the apparatus and methods forelectrode-based ion flow analyte monitoring through a nanomembranenanopore as provided herein. A sequence is derived from these data bycompiling the measured signals in comparison with the previouslydetermined signals correlated to a given DNA or RNA nucleotide, ordouble-strand base pairs, or modified versions thereof, as theysequentially pass through a protein nanopore residing in ananomembrane-stabilized lipid bilayer. A sequence also may be derived bymonitoring the timing at which measured signals change as a partiallydouble-stranded nucleic acids pass through such a protein nanopore. Asequence is derived from these data by compiling the measured signals incomparison with the signals previously correlated to single- versusdouble-stranded nucleic acids as they pass through a protein nanopore,knowledge of the sequence of the nucleic acid oligomer which was used toform the partial double-strands, and the timing between when the singleversus double strand nucleic acid signals were measured. In anotherembodiment, the primary electrode(s) upon which the lipidbilayer-supporting nanomembrane is deposited can be used to monitor thepassage of a nucleic acid through a given protein nanopore. Inparticular, the through-nanomembrane analyte monitoring apparatus andmethods described herein can be used for these embodiments. Regardlessof the signal monitoring method employed, though, nucleic acidsequencing can be carried out by compiling the measured signals of anucleic acid's passage through a given protein nanopore in comparisonwith previously correlated signals determined for single- and/ordouble-stranded nucleic acids, and/or modified versions thereof. Inother embodiments, the chloride/silver/silver chloride measuring systemdescribed herein can be utilized to monitor the flow of nucleic acidoligomers and chloride ions through a protein nanopore, and then derivethe sequence of the oligomer by comparison to pre-characterized signalsestablished for known nucleotides and/or groups of nucleotides.

In other embodiments, nanopores (i.e., nanomembrane-encirclednanochannels rather than one formed by a protein nanopore) can beutilized to enable nucleic acid sequencing. In these embodiments, asingle nanopore is formed within a given nanotube or nanowell. Nucleicacid oligomers are placed in the solution in the chamber(s) ‘above’ thenanopore. The nucleic acids can be single stranded DNAs or RNAsoligomers of any length. These DNAs and/or RNAs may or may not containnaturally or artificially modified nucleotides (e.g., naturally modifiedsuch as 5-methylcytidine; artificially modified such as to insertspacers between the nucleotides, and/or modified with ‘tags’ to increasetheir bulk, and/or bearing any other type of modified nucleic acid basessuch that they can be monitored by a given sensor system, etc). Thenucleic acids also may be double stranded, or partially double stranded,oligomers of any length. For partially double stranded DNAs, these canbe formed by hybridizing complementary nucleic acid oligomers of anylength so long as they are sufficiently stable to maintain theirpartially double-stranded forms in a useful manner for testing (e.g.,complementary oligomers of two or more nucleotides have been used inapplications in the art to make partially double-stranded DNAs). Thedouble-stranded or partially double-stranded DNA also may be naturallyor artificially modified as with single-stranded DNAs or RNAs. Afterplacing the nucleic acids ‘above’ the nanopore, an ion funnelcurrent/voltage can then be initiated to cause the negatively chargednucleic acids to move towards, and eventually through, the nanopore.FIGS. 4A and 4B summarize these processes, depicting the ion funnelcurrent/voltage-induced flow of single-stranded and partiallydouble-stranded nucleic acids through nanopores, respectively. Derivingthe sequence of the nucleic acid that is moving through a nanopore canbe carried out by several applications of the methods and apparatusdescribed herein. In one set of embodiments, the flow of ions or ionicmaterial passing through the nanopore along with a given nucleic acid ismonitored using the apparatus and methods described herein to monitorthe flow of ionic materials through nanomembrane nanochannels. Asequence is derived from these data by compiling the measured signals incomparison with the previously determined signals correlated to a givenDNA or RNA nucleotide, or double-strand base pairs, or modified versionsthereof, as they individually pass through a nanopore. A sequence alsomay be derived by monitoring the timing at which measured signals changeas a partially double-stranded nucleic acids pass through a givennanopore. A sequence is derived from these data by compiling themeasured signals in comparison with the signals previously correlated tosingle- versus double-stranded nucleic acids as they pass through agiven nanopore, knowledge of the sequence of the nucleic acid oligomerwhich was used to form the partial double-strands, and the timingbetween when the single- versus double-strand nucleic acid signals weremeasured. In another embodiment of the apparatus and method, the primaryelectrode(s) upon which the nanopore is deposited can be used to monitorthe passage of a nucleic acid through a given nanopore. In particular,the through-nanomembrane analyte monitoring apparatus and methodsdescribed herein can be useful for these embodiments. In someembodiments, one or more electrode above and below the general surfaceof the nanomembrane forming the nanopore can be used to monitor thepassage of the nucleic acid, which may or may not be accompanied byother ionized molecules, through the nanopore. Regardless of the signalgeneration method employed, nucleic acid sequencing is carried out bycompiling the measure signals of a nucleic acid's passage through agiven nanopore in comparison with previously correlated signalsdetermined for single- and/or double-stranded nucleic acids, and/ormodified versions thereof. In other embodiments, thechloride/silver/silver chloride measuring system described herein can beutilized to monitor the flow of nucleic acid oligomers and chloride ionsthrough a nanopore, and then derive the sequence of the oligomer bycomparison to pre-characterized signals established for knownnucleotides and/or groups of nucleotides.

In other embodiments, the nanomembrane portion of a given nanopore'souter perimeter can be functionalized via the attachment of, but notlimited to, proteins, enzymes, antibodies, lectins, peptides, aminoacids, modified amino acids, lipids, nucleic acids (single and doublestranded DNAs, RNAs, aptamers), nucleic acid components (nucleosides,nucleotides, methylated/modified versions of the same), ionized salts,small molecules, drugs, etc., and combinations thereof. In particular,it can be useful to manufacture the nanomembrane as described hereinsuch that the deposited material is in close proximity to where to thenanopore is positioned in a nanomembrane, and that this portion of thenanomembrane has chemical properties that are substantially differentfrom the bulk of the rest of the nanomembrane. Thus, such embodimentsprovide for functionalization attachment sites that can be highlylocalized to the nanopore's nanomembrane rim, and/or sidewall.

One non-limiting example of such embodiments is to add ‘target’molecules that can interact, be bound by, be chemically reacted upon by,etc. by an appropriate functionalization on the nanomembrane forming,and in particular in close proximity to, a nanopore. Targets herein aredefined as analytes that can be added over such a functionalizednanopore in solution and/or in gas phase and then specifically interactwith a given type of nanomembrane functionalization. Examples ofappropriate functionalizations and their ‘targets’ include, but are notlimited to: enzymes and their substrates; antibodies/antibody fragmentsand their antigens; protein drug targets and their drugs; aptamers andtheir ligands; lectins and their carbohydrate ligands; metalchelators/binders and metal ions; single-stranded DNA oligomers andtheir complementary DNA oligomers; and RNAs and RNA binding proteins.Measurements of the flow of ionic materials, targets and other analytesadded into the solution and/or gas placed ‘above’ a nanopore are thentaken over time, particularly under the conditions of the formation ofan ion funnel current/voltage as described herein, to monitor for signalchanges resulting from an interaction between a given form offunctionalization and its target. As one non-limiting example of this,FIGS. 5A and 5B depict the use of single-strand DNA-functionalizednanopore with an imposed ion funnel current/voltage, before and aftercomplementary single-strand DNA binding. As another non-limitingexample, FIGS. 6A and 6B depict the use of an antibody-functionalizednanopore with an imposed ion funnel current/voltage, before and afterantigen binding. The means by which the interaction of a given ‘target’and a nanopore's functionalization are monitored can include anyappropriate method, or combination of methods, described herein foranalyte flow monitoring. For example, embodiments to monitor for theresulting signal changes can include, but are not limited to, a decreasein the rate of flow of a given target through a nanopore, a decrease inthe flow of accompanying ionic material and/or chemically reactivespecies through a nanopore, and/or an increase in the rate of flow ofproducts resulting from an enzyme-catalyzed reaction through a nanopore.In other embodiments, the chloride/silver/silver chloride measuringsystem described herein can be utilized to monitor the flow of target(s)through the rim-functionalized nanopore, and then derive the sequence ofthe oligomer by comparison to pre-characterized signals established fora known target and/or groups of targets.

In other embodiments, nanomembranes are formed via methods describedherein such that a given nanopore is functionalized within itslateral-cross section (i.e., functionalization(s) are made to at leastsome portion of the encircling nanomembrane sidewall that forms a givennanopore). Examples of the methods for forming functionalizationattachment surfaces (including, in particular, ones in which theattachment surfaces that are restricted in close proximity to a nanoporesidewall's outer perimeter), modes of functionalization, andfunctionalization types are as included herein. In these embodiments, agiven ‘target’ of a functionalization type is provided in a solution orgas phase over the surface of a nanomembrane containing a nanopore. Themeans by which the interaction of this given ‘target’ and a nanopore'sfunctionalization is monitored can include any appropriate method, orcombination of methods, described herein for analyte monitoring. Forexample, embodiments to monitor for the resulting signal changes caninclude, but are not limited to, a decrease in the rate of flow of agiven target through a nanopore, a decrease in the flow of ions throughthe nanopore, and an increase in the rate of flow of products resultingfrom an enzyme-catalyzed reaction through a nanopore. In otherembodiments, the chloride/silver/silver chloride measuring systemdescribed herein can be utilized to monitor the flow of target(s)through the sidewall-functionalized nanopore, and then derive thesequence of the oligomer by comparison to pre-characterized signalsestablished for a known target and/or groups of targets.

In other embodiments, a nanopore can be functionalized with nucleases,enzymes capable of cleaving DNAs and or RNAs into nucleotides, with theoverarching application of enabling DNA and/or RNA oligomer sequencing.Examples of the methods for forming a nuclease attachment surface(including, in particular, ones in which the nuclease attachment surfaceis restricted in close proximity to a nanopore's outer perimeter), modesof nuclease functionalization, and nuclease functionalization types areas included herein. Both endo- and exonucleases can be utilized inembodiments, as well as can nucleases with specificities includingsingle- and/or double-stranded DNAs and/or single-stranded RNAs and/ormodified versions of the same. Nucleic acid oligomers can be single-and/or double-stranded DNAs, single-stranded RNAs, modified versions ofthe same as described herein, and can be oligomers of any length. Forsuch embodiments, an ion funnel current/voltage is initiated and nucleicacid oligomers are placed in solution in the chamber(s) ‘above’ anuclease-functionalized nanopore (FIGS. 3A-3C). The ion funnelcurrent/voltage then causes the negatively charged nucleic acidoligomers to move towards a nanopore where one of the nucleases attachedat its rim sequentially cleaves the oligomer into nucleotides (FIG. 3D).The nucleotides, which also are negatively charged, are drawninto/through the nanopore due the effects of the ion funnelcurrent/voltage. Deriving the sequences of the nucleic acid oligomerscan be carried out by several applications of the methods and apparatusdescribed herein. Whatever mode is used to identify the oligomer'ssequence, though, the overarching process of obtaining an oligomerssequence by identifying the nucleotides that result from an oligomer'scleavage is generally described in the art as‘sequencing-by-degradation.’ In one set of embodiments, the nucleotidesare directly sampled and the identified from material that issequentially taken from the space below the nanopore (e.g., from samplestaken from the space(s) ‘below’ the nanopore that are in the nanotube ornanowell in which they are formed and/or the chamber(s) below thenanotube's exit from its solid support). In another set of embodiments,the flow of the nuclease-cleaved nucleotides ions passing through thenanopore is monitored using the apparatus and methods for ionic analytemovement monitoring over nanomembranes as provided herein. For example,in embodiments, one or more electrodes above and below the generalsurface of the nanomembrane forming the nanopore are used to monitor thepassage of the ionized nucleotides, and/or the flow of other ionizedanalytes that can be in motion with the nucleotides, which may or maynot be accompanied by other ionized molecules, through the nanopore. Asequence is derived from these data by compiling the measured signals ofthe moving ionized nucleotides, and/or other ionized molecules in motionwith the nucleotides, in comparison with the previously determinedsignals correlated to a given DNA or RNA nucleotide. In anotherembodiments, the primary electrode(s) upon which a given nanopore isdeposited can be used to monitor the passage of nucleotides through agiven nanopore. In particular, the through-nanomembrane analytemonitoring apparatus and methods appropriate for monitoring nucleotidesand as described herein can be particularly useful for theseembodiments. In particular, it can be useful to employ methods in whichelectrical stimuli is applied to an electrically conductive nanomembraneand then measurements such as capacitance, current, voltage, etc. aretaken at one or more secondary electrodes that form one or more otherportions of a nanopore's cross-section, or are taken at one or moresecondary electrodes above or below the nanopore's cross-section. In anycase, nucleic acid oligomer sequences are derived by compiling themeasure signals of the nucleotides' passage through a given nanopore incomparison with previously correlated signals determined for single-and/or double-stranded nucleotides, and/or modified versions thereof. Inother embodiments, the chloride/silver/silver chloride measuring systemdescribed herein can be utilized to monitor the flow of nucleotides andchloride ions through a nanopore, and then derive the sequence of theoligomer by comparison to pre-characterized signals established forknown nucleotides and/or groups of nucleotides.

In embodiments, a suitable nanochannel within a nanomembrane (i.e., ananochannel that is not entirely encircled by nanomembrane as is definedherein as being a nanopore) can be used in place of a nanopore for manythe applications just described. Suitable nanochannels can include, butare not limited to, ones that provide for functionalization sites, ifrequired, that can be highly localized to nanochannel's nanomembranerim. Nanochannels also can include ones that have at least some portionof their sidewall defined by the placement of a nanomembrane(s) suchthat a required through-nanomembrane monitoring capabilities can beenabled. Nanochannels that are nearly encircled by depositednanomembrane also can be useful since the electroactivity of thenanomembrane can be employed as required to tunably decrease thecross-section area through which a given analyte moves. Lowering thecross-section area has been described as helping to improve thesignal-to-noise monitoring of analytes like nucleic acids as they movethrough protein nanopores (Clark et al., Nat. Nanotechnol 2009).

In embodiments, an imposed ion funnel current/voltage is applied to asolution containing double-stranded DNA that has been placed over anuclease-functionalized nanopore or suitable nanochannel. However,instead of the nucleotides being drawn through the nanopore to enablethe sequence determinations, the single-stranded DNA that is the otherproduct of the nuclease reaction is caused to ‘thread’ through thenanopore or suitable nanochannel. The modes for sequencing thesingle-stranded DNA oligomer as it threads through a nanopore orsuitable nanochannel are as is described previous embodiments.

Note that conventional sequencing-by-synthesis modes only provide forquite short oligomer sequence read lengths. For example, currentcommercialized high-throughput DNA sequencing technologies that employ aDNA polymerase or DNA ligase to generate a sequencing signal byelongating the copy-strand of a DNA oligomer rarely provided accurateread lengths in excess of a few hundred bases. Such short read lengthsgreatly complicate the speed, accuracy, and cost of aligning theresulting data to provide for a high accuracy full-genome sequence map.The disclosed apparatus and methods provided herein for both thesequencing-by-threading and sequencing-by-degradation modes solve thisproblem. For example, the read lengths that can be derived from thesequencing-by-threading process described herein can be as long as thethreaded oligomers themselves. Since it easily is possible to preparesamples of nucleic acid oligomers in excess of tens, or even hundreds ofthousands, of bases in length, it can be possible to obtain read lengthsthat are orders of magnitude greater than is possible forsequencing-by-synthesis processes. Similarly, for the sequencingapparatus and methods mediated via sequencing-by-degradation describedherein, the nuclease cleavage reactions underlying theses technologiesare well known to be highly processive in that they generally continueto sequentially cleave a given nucleic acid oligomer until the end ofthe oligomer is reached, and regardless of the oligomers length. Thus,the sequencing-by-degradation apparatus and methods described hereintherefore can provide read lengths that may be limited only by thelengths of the oligomers placed in a given sequencing sample. Hence, theapparatus and methods provided herein for sequencing-by-degradation canprovide read lengths that are orders of magnitudes greater than what ispossible for current commercialized sequencing-by-degradationtechnologies.

Furthermore, the apparatus and methods provided herein provide directsequencing-by-threading and/or sequencing-by-degradation of RNAoligomers in modes that do not necessarily require the use of reversetranscriptases to convert these RNA oligomers into cDNAs. Reversetranscriptases are well known to introduce errors and increase the costfor RNA sequencing. In addition, it can be difficult and costly tosequence biologically important but inherently short oligomer RNAoligomers like those of the miRNAs via current sequencing methods. Thus,the apparatus and methods provided herein can greatly decrease the costand increase the accuracy of RNA oligomer sequencing.

FIGS. 3A-3D depict analyte motion-inducing methods using devices ofFIGS. 1 and/or 2A through 2C according to various embodiments of thepresent teachings. The analyte 366 can be induced to move across a givennanowell (see FIG. 1) and/or a given nanomembrane deposited within ananowell (see FIGS. 2A-2C, or FIG. 3A) by subjecting a passive Brownianmotion-driven diffusion movements, an electrophoretic force, and/or amechanical pressure. In an exemplary embodiment, imposed electricalstimuli create an “ion funnel current/voltage” 370 to move the analyte366.

FIGS. 4A-4B depict an exemplary embodiment of nucleic acid sequencingfor a single-stranded nucleic acid 466 or a single-stranded nucleic acid466 with partially double-stranded nucleic acid 469 through a nanoporeor a suitable nanochannel. DNA and RNA threading through nanopore orsuitable nanochannel can be conducted by positional sequencing usingpartially double-stranded DNAs. FIGS. 5A-5B depict an exemplaryembodiment in monitoring DNA hybridization; while FIGS. 6A-6B depict anexemplary embodiment in monitoring antigens binding to antibodies.

Employing that the nanotechnologies and their compatibility to standardsilicon manufacturing processes, the present disclosure makes itpossible to form cost-effective handheld analyte detectors and/or tocarry out parallel analyte detection reactions in a server blade-styleformat such that scaling for two or more simultaneous independentdetection can be carried out. The analytes detected need not be the samein such parallel operations. While during and/or after a given analytedetection reaction is carried out, all of the resulting data can becommunicated by wires (standards such as ethernet, usb, or dial-up) orwirelessly (standards such as zigbee, Bluetooth, wifi, WiMax, citizensband, satellite link, or cell phone formats) or combinations thereof(standards such as wireless connection to land line-based communicationslinks).

As disclosed, nanopores and/or nanochannels formed by electroactivenanomembrane(s) can have an electrically tunable diameter as a resultfrom an electroactive response of the nanomembranes. In embodiments,insulating layers, such as for example, a metal oxide, glasses,nonconductive polymers, and/or silicon, can be included to form thenanopores/nanochannels. For example, alternating layers of anelectroactive nanomembrane and an insulating material can be formed onat least one electrode of a nanotube or a nanowell. The alternatinglayers can be formed by, e.g., the disclosed electrochemical deposition,and other methods such as energetic neutral beam lithography/epitaxy. Insome embodiments, a place holder or a template can be placed within acorresponding nanotube or nanowell during deposition of nanomembranesand then removed from the deposited nanomembranes leaving a nanopore ora nanochannel formed by the deposited one or more nanomembranes. Forexample, the place holder/template can be a cylinder or a strip formedby, e.g., photocurable polymer. In another example, the insulating layercan be deposited over the electroactive nanomembrane but expose an edgeof the nanomembrane for further deposition of, e.g., a polymer, on theedge. In other embodiments, nanopores, nanochannels can be formed byfirst depositing nanomembranes to fill nanotubes/nanowells and thendrilling openings through the nanomembranes, e.g., using a focused ionbeam.

In embodiments, nanopores or nanochannels can be used for treating ananalyte molecule or control flow of a fluid. For example, a sensorstructure or a flow controlling structure can be formed by using aseparation structure, which can be the disclosed device includingnanomembranes deposited in nanowells/nanotubes in a support material.The separation structure can separate a sample chamber from a collectionchamber such that analyte molecules or other molecules or the fluid canmove from the sample chamber through nanopores/nanochannels and into thecollection chamber. The sensor structure can further include a firstelectrode pair having electrodes disposed at opposite ends (e.g.,including the bottom electrode) of the electroactivenanopore/nanochannel. The sensor structure can also include a secondelectrode pair (e.g., including sidewall electrodes) disposed in thesidewall of nanowells/nanotubes between the first electrode pair. Thenanomembranes can be disposed over at least one electrode of the secondelectrode pair.

In operation, a first electrical stimuli that are sufficient to causethe analyte molecule or other molecules or the fluid to migrate from thesampling chamber through the electroactive nanopores/nanochannels to thecollection chamber can be applied across the first electrode pair. Whenanalyte molecules or the fluid in the sampling chamber pass through theelectroactive nanopores/nanochannels, a current across the firstelectrode pair, e.g., indicative of the presence of the analytemolecule, can be measured. The diameter of the nanopores/nanochannelsformed by the electroactive membrane(s) can be electrically tuned (i.e.,increased or decreased) by applying a second electrical stimuli acrossthe second electrode pair, which causes the electroactive membrane(s) toeither expand or contract. In embodiments, a flow of an ionic speciescan also be controlled through the nanopores/nanochannels, e.g., byattracting the ionic species using an ion funnel current/voltage throughthe opening of the nanopores/nanochannels and tuning (increasing ordecreasing) the electrically tunable diameter of the opening so as tocontrol flow of the ionic species through the nanopores/nanochannels.This application discloses several numerical range limitations thatsupport any range within the disclosed numerical ranges even though aprecise range limitation is not stated verbatim in the specificationbecause the embodiments of the invention could be practiced throughoutthe disclosed numerical ranges. Finally, the entire disclosure of thepatents and publications referred in this application, if any, arehereby incorporated herein in entirety by reference.

1. A method comprising: providing a nanochannel within a supportmaterial wherein the nanochannei comprises one or more sidewallelectrodes; disposing a nanomembrane inside the nanochannel, wherein thenanomembrane is configured to encircle a nanopore, has a hydrophobicsurface, and is in direct contact with at least a portion of onesidewall electrode of the nanochannel; tuning a size of the nanopore bytuning dimensionality of the nanomembrane with an electrical stimulus onthe nanomembrane: tethering a hydrophobic material on the hydrophobicsurface, the hydrophobic material spanning the nanonore; disposing aprotein nanopore into the hydrophobic material, wherein the size of thenanopore is tuned such that the hydrophobic material spanning thenanopore is configured to prevent more than one protein nanopore.
 2. Themethod of claim 1, further comprising stabilizing the hvdrophobicmaterial.
 3. The method of claim 1, wherein the protein nanoporecomprises an α-hemoivsin or a mycobacterium smegmatis porin A.
 4. Themethod of claim 1, further comprising disrupting the hydrophobicmaterial by attracting, repulsing, or electrophoresing charged moleculesto or from a lipid bilayer, by an electromotive force on the hydrophobicmaterial or by a temperature change.
 5. The method of claim 1, whereinforming the nanomembrane is by the method selected from a groupconsisting of spraying, vapor-phase deposition, sputtering, spincoating, precipitation, in situ polymerization, and a combinationthereof.
 6. The method of claim 1, wherein a mechanical, a chemical orelectrical property of the nanomembrane is uniform.
 7. The method ofclaim 1, wherein the nanochannel further comprises one or more bottomelectrodes exposed to the nanochannel.
 8. The method of claim 1, whereinthe nanomembrane comprises two layers of distinct properties.
 9. Themethod of claim 1, wherein the electrical stimulus comprises anelectrical current, a voltage, or an electrical waveform.
 10. The methodof claim 1, wherein at least a portion of the nanomembrane iselectrically conductive.
 11. The method of claim 1, wherein a DNA or RNAmolecule is threaded through the protein nanopore.
 12. The method ofclaim 11, wherein the DNA or RNA molecule is threaded through theprotein nanopore via direct or indirect electrophoretic forces.
 13. Themethod of claim 11, wherein the DNA or RNA molecule is threaded throughthe protein nanopore via direct or indirect mechanical forces, includingflow of a liquid or active protein-induced threading of the DNA or RNAmolecule.
 14. The method of claim 1, wherein signals are taken via thebottom or side wall electrodes in order to sequence a DNA or RNAmolecule as the DNA or RNA molecule is being threaded through theprotein nanopore.
 15. The method of claim 14, wherein the signals aretaken via the bottom or side wall electrodes in order to identifymodified DNA or RNA bases on the DNA or RNA molecule as the DNA or RNAmolecule is being threaded through the protein nanopore.
 16. The methodof claim 1, wherein the hydrophobic material comprises, or is in part, agel.
 17. The method of claim 16, wherein the gel comprises a responsivegel.